U.S. patent number 7,227,002 [Application Number 09/403,107] was granted by the patent office on 2007-06-05 for human antibodies that bind human 17-a1/epcam tumor antigen.
This patent grant is currently assigned to Micromet AG. Invention is credited to Peter Kufer, Tobias Raum.
United States Patent |
7,227,002 |
Kufer , et al. |
June 5, 2007 |
Human antibodies that bind human 17-A1/EpCAM tumor antigen
Abstract
The present invention provides an anti-human antibody or
fragment thereof that is low or not immunogenic in humans. In
particular, the antibodies or fragments are directed to human tumor
antigens, preferably to the human tumor antigen 17-1A, also known
as EpCAM, EGP or GA 733-2. Also provided are pharmaceutical
compositions comprising the aforementioned antibodies or fragments
thereto.
Inventors: |
Kufer; Peter (Moosburg,
DE), Raum; Tobias (Munich, DE) |
Assignee: |
Micromet AG (Munich,
DE)
|
Family
ID: |
8226694 |
Appl.
No.: |
09/403,107 |
Filed: |
April 14, 1998 |
PCT
Filed: |
April 14, 1998 |
PCT No.: |
PCT/EP98/02180 |
371(c)(1),(2),(4) Date: |
October 14, 1999 |
PCT
Pub. No.: |
WO98/46645 |
PCT
Pub. Date: |
October 22, 1998 |
Foreign Application Priority Data
|
|
|
|
|
Apr 14, 1997 [EP] |
|
|
97106109 |
|
Current U.S.
Class: |
530/387.3;
424/156.1; 424/142.1; 424/174.1; 424/183.1; 530/388.15; 530/388.85;
530/391.3; 530/391.7; 536/23.53; 435/69.6; 424/181.1;
424/133.1 |
Current CPC
Class: |
A61P
37/00 (20180101); C07K 16/00 (20130101); A61P
37/04 (20180101); A61P 35/00 (20180101); C07K
16/30 (20130101); C07K 2317/34 (20130101); A61K
38/00 (20130101) |
Current International
Class: |
C12P
21/08 (20060101); C07K 16/00 (20060101); A61K
39/395 (20060101); C07H 21/04 (20060101); C12P
21/04 (20060101) |
Field of
Search: |
;530/300,350,387.1,387.3,388.1,388.15,388.8,388.85,391.3,391.7
;424/130.1,133.1,139.1,135.1,142.1,156.1,179.1,180.1,183.1 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
5565332 |
October 1996 |
Hoogenboom et al. |
5885793 |
March 1999 |
Griffiths et al. |
6150584 |
November 2000 |
Kucherlapati et al. |
|
Foreign Patent Documents
|
|
|
|
|
|
|
WO 93/11236 |
|
Jun 1993 |
|
WO |
|
WO 97/00271 |
|
Jan 1997 |
|
WO |
|
Other References
Rudikoff et al (Proc Natl Acad Sci USA 1982 vol. 79 p. 1979). cited
by examiner .
Freshney (Culture of Animal Cells, A Manual of Basic Technique,
Alan R. Liss, Inc., 1983, New York, p. 4. cited by examiner .
Dermer (Bio/Technology, 1994, 12:320). cited by examiner .
Gura (Science, 1997, 278:1041-1042). cited by examiner .
Jain (Sci. Am., 1994, 271:58-65). cited by examiner .
William E. Paul. Fundamental Immunology, 3rd edition, pp. 292-295,
1993. cited by examiner .
Albert F. LoBuglio et al., Proc. Natl. Acad. Sci, 86:4220-4224,
1989. cited by other .
Duenas et al.: In vitro immunization of naive human B cell yields
high affinity immunoglubulin G antibodies as illustrated by phage
display. IMMUNOLOGY, vol. 89, No. 1, Sep. 1996, pp. 1-7,
XP002093905 Oxford, GB. cited by other .
A. Krebber et al.: "Reliable cloning of functional antibody
variable domains from hybridomas and spleen cell repertoire
employing a reengineered phage display system", Journal of
Immunological Methods, vol. 201, No. 1, Feb. 14, 1997, pp. 35-35,
XP002093906 Amsterdam, The Netherlands. cited by other .
H. Gottlinger et al.: "The epithelial cell surface antigen 17-1A, a
target for antibody-medicated tumor therapy: its biochemical
nature, tissue distribution and recognition by different monoclonal
antibodies" International Journal of Cancer, vol. 38, No. 1, Jul.
15, 1986, pp. 47-53, XP002093908 New York, NY, USA. cited by other
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A. Hoess et al.: "Generation of human antibodies that selectively
recognize diseased cells overexpressing surface bound antigens."
Proceedings of the American Association for Cancer Research vol.
38, Mar. 1997, p. 30 XP002093904 USA, Abstract #198. cited by other
.
Figini et al., "In vitro assembly of repertoires of antibody chains
on the surface of phage by renaturation," J. Mol. Biol., 239:68-78,
1994. cited by other .
Marks et al., "By-passing immunization human antibodies from V-gene
libraries displayed on phage," J. Mol. Biol., 222:581-597, 1991.
cited by other .
U.S. Appl. No. 10/325,694, filed Dec. 19, 2002, Kufer. cited by
other .
Herlyn et al. "Colorectal carcinoma-specific antigen: Detection by
means of monoclonal antibodies," Proc. Natl. Acad. Sci.,
76:1438-1442, 1979. cited by other .
Herlyn et al., "IgG2a monoclonal antibodies inhibit human tumor
growth through interaction with effector cells," Proc. Natl. Acad.
Sci., 79:4761-4765, 1982. cited by other .
Litvinov et al., "Ep-CAM: a human epithelial antigen is a
homophilic cell-cell adhesion molecule," Department of Pathology,
State University of Leiden, Leiden, The Netherlands, 125:437-446,
1994. cited by other .
Sun et al., "Chimeric antibody with human constant regions and
mouse variable regions directed against carcinoma-associated
antigen 17-1A," Proc. Natl. Acad. Sci., 84:214-218, 1987. cited by
other.
|
Primary Examiner: Blanchard; David J.
Attorney, Agent or Firm: Fulbright & Jaworski
Claims
The invention claimed is:
1. An isolated antibody or antibody fragment being low or not
immunogenic in humans and recognizing the human 17-1A antigen as
expressed on the surface of tumor cells, the antibody or antibody
fragment being further characterized as comprising a human VH chain
and a human VL chain wherein at least said VH chain is a VH
sequence of unprimed mature human B-lymphocytes and comprises three
CDRs encoded by nucleotides 91 to 105, 148 to 198 and 292 to 351
within nucleotides 1 to 381 of SEQ ID NO: 143 and said VL chain is
a VL sequence of a naturally occurring human B cell repertoire and
comprises three CDRs encoded by nucleotides 70 to 102, 148 to 168
and 265 to 294 within nucleotides 1 to 321 of SEQ ID NO: 141.
2. The antibody or antibody fragment according to claim 1, which is
an antibody.
3. The antibody or antibody fragment of claim 1, recognizing an
epitope of the extracellular domain of the 17-1A antigen.
4. The antibody or antibody fragment of claim 1, wherein said human
VH chain comprises the amino acid sequence encoded by nucleotides 1
to 381 of SEQ ID NO: 143 and/or said human VL chain comprises the
amino acid sequence encoded by nucleotides 1 321 of SEQ ID
NO:141.
5. The antibody or antibody fragment according to claim 1, further
comprising fused to said human VH and VL chains, immunoglobulin
constant regions of heavy (CH) and light chains (CL), respectively,
or non-immunoglobulin chains.
6. The antibody or antibody fragment according to claim 5, wherein
said constant region chains are from human IgG1 or IgG3.
7. The antibody or antibody fragment according to claim 1, further
comprising a radioisotope, chemotherapeutic agent or toxin linked
to said human VH and VL chains.
8. A pharmaceutical composition comprising an antibody or antibody
fragment according to claim 1, 5, 6 or 7, and a pharmaceutically
acceptable carrier.
9. A pharmaceutical composition comprising an antibody or antibody
fragment according to claim 3, and a pharmaceutically acceptable
carrier.
10. A pharmaceutical composition comprising an antibody or antibody
fragment according to claim 4, and a pharmaceutically acceptable
carrier.
Description
This application is a U.S. National Phase Application Under 35
U.S.C. .sctn. 371 of International Application No. PCT/EP98/02180
filed Apr. 14, 1998, which claims priority to European Application
No. 971061098 filed Apr. 14, 1997.
The present invention relates to a method for the production of an
anti-human antigen receptor that is low or not immunogenic in
humans comprising the steps of selecting a combination of
functionally rearranged VH and VL immunoglobulin chains wherein at
least said VH chain is derived from essentially unprimed mature
human B-lymphocytes or from essentially anergic human B cells and
said VL chain is derived from a naturally occurring human B cell
repertoire, said chains being expressed from a recombinant vector
and using an in vitro display system for binding to a human
antigen.
The present invention further relates to receptors that are low or
not immunogenic in humans and directed to human antigens, said
receptors being obtainable by the method of the invention. Said
receptors are preferably antibodies or fragments thereof or
immunoconjugates comprising the VH/VL chains of said antibody. In
particular, the receptors of the invention are directed to human
tumor antigens, preferably to the human tumor antigen 17-1A, also
known as EpCAM, EGP-40 or GA 733-2. Finally, the present invention
relates to kits useful for carrying out the method of the
invention.
The mammalian immune systems such as the human immune system select
against immune competent cells and molecules that are specific for
self-antigens. Dysregulation of the immune system in this regard
may result in auto-immune diseases such as rheumatoid arthritis. In
general, the surveillance of the immune system with regard to the
autoreactive antibodies or T cells is therefore highly beneficial.
However, there may be cases where it would be advantageous to have
autoreactive antibodies that are directed to antigens expressed in
the mammalian, and in particular, the human body. Such antigens
are, for example, tumor associated antigens. For example, the human
17-1A or EpCAM antigen, a surface glycoprotein expressed by cells
of simple epithelia and malignant tumors derived thereof, has been
shown to be a rewarding target for monoclonal antibody therapy of
cancer, especially in patients with minimal residual disease
suffering from disseminated tumor cells that may cause later solid
metastasis and thus impairs patients' prognosis. In patients with
minimal residual colorectal cancer, a murine monoclonal antibody
specific for the human 17-1A-antigen decreased the 5-year mortality
rate by 30% compared to untreated patients, when applied
systemically in five doses within four months after surgery of the
primary tumor; in total each patient was treated with 900 mg of
antibody (Riethmuller, Lancet 343 (1994), 1177 1183). However,
during the course of antibody treatment patients developed a strong
antibody response against the murine immunoglobulin. These human
anti-mouse antibodies (HAMAs) severely limit the continuous
application of antibody therapies and increasingly reduce their
efficacy. Moreover, preformed HAMAs induced by former antibody
treatment or another contact with murine immunoglobulin can
severely interfere with later antibody therapies.
To prevent these problems, therapeutic antibodies with minimal
immunogenicity would be preferable. To achieve this goal, it might
be, for example, envisaged that therapeutic antibodies or antibody
derivatives are completely human by their amino acid sequence and
the immunogenic profile of the human antibody idiotype is minimized
by using human Ig-variable regions likely to be tolerated by the
human immune system.
However, the generation of human antibodies against human antigens
faces two major problems: (1) Hybridoma or other cell
immortalisation techniques proved to be quite inefficient in
generating human antibody producing cell lines compared to the
murine hybridoma technology. (2) Auto-reactive antibodies are
relatively efficiently depleted of naturally occurring antibody
repertoires due to the mechanisms mediating self-tolerance.
Human antibodies in general have become much more accessible since
the availability of transgenic mice expressing human antibodies
(Bruggemann, Immunol. Today 17 (1996), 391 397) and of the
combinatorial antibody library and phage display technology
allowing the in vitro combination of variable regions of Ig-heavy
and light chains (VH and VL) and the in vitro selection of their
antigen binding specificity (Winter, Annu. Rev. Immunol. 12 (1994),
433 455). By using the phage display method, rare events like one
specific binding entity out of 10.sup.7 to 10.sup.9 different
VL/VH- or VH/VL-pairs can easily be isolated; this is especially
true when the repertoire of variable regions has been enriched for
specific binding entities by using B-lymphocytes from immunized
hosts as a source for repertoire cloning.
Often, however, the frequency of specific binding entities is
substantially lowered in naturally occurring antibody repertoires.
This is particularly true for cases of antibodies binding to
self-antigens. Random combinations of VL- and VH-regions from a
self-tolerant host resulting in a combinatorial antibody library of
conventional size (10.sup.7 to 10.sup.9 independent clones) most
often are not sufficient for the successful in vitro selection of
auto-reactive antibodies by the phage display method.
One strategy to circumvent this problem is the use of very large
combinatorial antibody libraries that compensate by the library
size for the low frequency of auto-reactive antibodies in naturally
occurring repertoires. Combinatorial antibody libraries exceeding a
size of 10.sup.9 independent clones, however, are difficult to
obtain because of the current technical limit of the transformation
efficiency for plasmid-DNA into E. coli-cells.
To avoid the self-tolerance mediated bias in naturally occurring
antibody repertoires, that underrepresents auto-reactive antibodies
and markedly decreases the chances of isolating antibodies
specifically recognizing self-antigens, approaches using
semisynthetic or fully synthetic VH- and/or VL-chain repertoires
have been developed. For example, almost the complete repertoire of
unrearranged human V-gene-segments has been cloned from genomic DNA
and used for in vitro recombination of functional variable region
genes, resembling V-J- or V-D-J-recombination in vivo (Hoogenboom,
J. Mol. Biol. 227 (1992), 381 388; Nissim, EMBO J. 13 (1994) 692
698; Griffiths, EMBO J. 13 (1994), 3245 3260). Usually, the
V-D-/D-J-junctional and the D-segment diversity mainly responsible
for the extraordinary length and sequence variability of heavy
chain CDR3 as well as the V-J-junctional diversity contributing to
the sequence variability of light chain CDR3 is imitated by random
sequences using degenerated oligonucleotides in fully synthetic and
semisynthetic approaches (Hoogenboom (1994), supra; Nissim, supra;
Griffiths, supra; Barbas, Proc. Natl. Acad. Sci. U.S.A. 89 (1992),
44574461).
However, VL/VH- or VH/VL-pairs selected for binding to a human
antigen from such semisynthetic or fully synthetic repertoires
based on human V-gene sequences are at risk of forming immunogenic
epitopes that may induce an undesired immune response in humans
(Hoogenboom, TIBTECH 15 (1997), 62 70); especially the CDR3-regions
derived from completely randomized sequence repertoires are
predestined to form potentially immunogenic epitopes as they have
never had to stand the human immune surveillance without being
recognized as a foreign antigen resulting in subsequent
elimination. This is equally true for human antibodies from
transgenic mice expressing human antibodies as these immunoglobulin
molecules have been selected for being tolerated by the murine but
not the human immune system.
Accordingly, the technical problem underlying the present invention
was to provide a method that allows the production of receptors
that are low or not immunogenic in humans and that can be used for
targeting antigens in the human body. The solution to said
technical problem is achieved by providing the embodiments
characterized in the claims.
Thus, the present invention relates to a method for the production
of an anti-human antigen receptor that is low or not immunogenic in
humans comprising the steps of selecting a combination of
functionally rearranged VH and VL immunoglobulin chains wherein at
least said VH chain is derived from essentially unprimed mature
human B-lymphocytes or from essentially anergic human B cells and
said VL chain is derived from a naturally occurring B cell
repertoire, said chains being expressed from a recombinant vector
and using an in vitro display system for binding to a human
antigen.
The method of the present invention is highly advantageous for
providing receptors that can be used for targeting antigens in
humans without being at all or to any significant extent
immunogenic themselves. Such receptors can advantageously be used
for treating a variety of diseases such as tumors or auto-immune
diseases, graft rejection after transplantation, infectious
diseases by targeting cellular receptors as well as allergic,
inflammatory, endocrine and degenerative diseases by targeting key
molecules involved in the pathological process.
The VH/VL immunoglobulin chains of the receptors of the present
invention can, of course, be further investigated with regard to
their nucleic acid and amino acid sequences using techniques
well-known in the art, see e.g. Sambrook (Molecular Cloning; A
Laboratory Manual, 2nd Edition, Cold Spring Harbour Laboratory
Press, Cold Spring Harbor, N.Y. (1989)). Once the nucleic acid
sequence or the amino acid sequence have been determined, the
receptors of the invention can also be produced by other methods,
such as by synthetic or semi-synthetic methods yielding synthetic
or semi-synthetic receptors, or in transgenic mice expressing human
immunoglobulin receptors; carrying the features recited herein
above and produced by such synthetic or semi-synthetic methods or
in said transgenic mice are also included within the scope of the
present invention.
After binding of the receptor to the human antigen, the receptor
can be further purified. For example, non-bound receptors which do
not carry the antigen specificity may be removed by washing steps.
The bound receptor may be eluted from the human antigen and further
purified, wherein additional rounds of expression, binding and
selection of the desired receptor may be used until the desired
receptor and/or the corresponding recombinant vector have been
isolated in pure form.
The method of the present invention thus makes use of the
preselection of Ig-variable regions by the human immune system. The
receptors are derived from a repertoire selected in vitro from
human combinatorial antibody libraries exclusively or
preferentially made of the naturally occurring antibody repertoire
expressed by essentially unprimed mature human B-lymphocytes or
from essentially anergic human B-cells.
However, the Ig variable domains may also be derived from a variety
of other sources that represent these preselected populations of B
cells.
The scientific background with regard to the origin of the B cells
functioning as a source of said VH or VL chains, may be explained
as follows:
Mature unprimed B-lymphocytes, expressing IgD and IgM as membrane
antigen receptors enter primary follicles during their traffic to
and between secondary lymphoid organs unless they have encountered
multivalent self antigen resulting in clonal deletion or soluble
monovalent self antigen rendering them anergic and short-lived due
to exclusion from primary follicles.
Contact of immature B-cells, that exclusively express IgM, with
self antigen in the bone marrow results in clonal deletion or
anergy depending on the antigen valency. Anergic B-cells, although
expressing surface IgD, are unable to respond to the antigen
through this receptor; without access to primary follicles and in
the absence of T-cell help, these cells have a short half-life of
only a few days.
In contrast, mature unprimed B-lymphocytes that have not encounted
self antigen and therefore have access to primary follicles have a
long half-life of several weeks. Despite the described mechanisms
mediating self-tolerance, these populations of long-lived mature
unprimed B-lymphocytes still contain potentially self-reactive
B-cells, that are, however, unlikely to find specific T-cell help
due to T-cell tolerance and thus kept from proliferation and
antibody secretion. It appears that B-cell non-responsiveness to
many self-antigens that are present at low levels is of this type,
affecting the helper T-cells but not the B-cells. In the present
invention these long-lived, non-responsive, potentially
self-reactive mature unprimed B-lymphocytes have been identified as
the most promising naturally occurring human antibody repertoire
for constructing combinatorial antibody libraries especially suited
to select human antibodies to human antigens by, for example, the
phage display method.
This highly selected antibody repertoire used as a basis for the
present invention mainly derived from B-cells with a long in vivo
half-life and thus exposed to the human immune system for prolonged
periods of time is expected to be markedly depleted of antibody
molecules forming epitopes especially within the highly variable
CDR3-regions, that are immunogenic for the human immune system.
Therefore, human antibodies selected from this antibody repertoire
are expected to have a lower immunogenic profile in humans than
human antibodies selected from semisynthetic or fully synthetic
human antibody libraries.
Mature unprimed B-cells that are activated by contact with foreign
antigen stop to express IgD and start clonal proliferation and
differentiation into plasma cells secreting soluble immunoglobulin;
early stages of the antibody response are dominated by
IgM-antibodies, while later, IgG and IgA are the predominant
isotypes, with IgE contributing a small but biologically important
part of the antibody response.
Unlike IgD-negative mature antigen-primed B-lymphocytes expressing
IgM, IgG, IgA or IgE, IgD-positive mature unprimed B-cells have not
yet undergone clonal proliferation, so that combinatorial
IgD-libraries do not overrepresent antibody specificities that are
currently or have been formerly involved in immune responses
usually driven by foreign antigen, thus decreasing repertoire
diversity and wasting library space for antibody candidates
unlikely to bind self antigen. This is in clear contrast to the
prior art recommending the use of human IgM combinatorial antibody
libraries for the in vitro selection of human antibodies against
human antigens from naturally occurring human antibody repertoires
(Hoogenboom (1997), supra).
In a preferred embodiment of the method of the invention, said
antigen receptor is an immunoglobulin or a fragment thereof.
The fragment of the immunoglobulin may be any fragment that is
conventionally known in the art such as Fab or F(ab).sub.2
fragments.
In a particularly preferred embodiment of the method of the
invention, said immunoglobulin fragment is an Fv-fragment.
In a further preferred embodiment of the method of the invention,
at least said VH and optionally said VL immunoglobulin chain are
derived from a human IgD repertoire.
This receptor and preferably antibody repertoire selected for low
immunogenicity has been concluded to be best represented in a human
IgD-antibody library. IgD is expressed as membrane antigen receptor
together with surface IgM on mature unprimed B-lymphocytes that
enter primary follicles during their traffic to and between
secondary lymphoid organs unless they have encountered multivalent
self antigen resulting in clonal deletion or soluble monovalent
self antigen rendering them anergic and short lived due to
exclusion form primary follicles. In addition to the antibody
repertoire of mature unprimed B-lymphocytes, human IgD-libraries
only further represent that of short-lived B-cells that have been
rendered anergic in contact with soluble monovalent self antigen
but are unlikely to contribute specific binders to human cell
surface molecules resembling multivalent self-antigens that induce
clonal deletion instead of B-cell anergy.
In a further preferred embodiment of the method of the invention,
said in vitro display system is a phage display system.
The phage display system has, in the past, conveniently been used
for the selection of a variety of peptides and proteins that bind
to specific targets. On the basis of this knowledge, the
immunoglobulin VH and VL chains can conveniently be cloned into
vectors that also comprise molecules useful for phage display
systems. Such molecules and vectors, respectively, are well-known
in the art (Winter, supra; Barbas, METHODS: A Companion to Methods
in Enzymology 2 (1991), 119 124) and need not be explained here in
more detail.
In a further preferred embodiment of the method of the invention,
said combination of rearranged chains is expressed from one or more
different libraries.
This embodiment is particularly preferred, if a VH or VL chain is
known that binds to a specific target and the corresponding second
V chain that reconstitutes or improves binding is selected.
In a further preferred embodiment of the method of the invention,
said human antigen is a tumor antigen.
If the human antigen is a tumor antigen, said antigen is preferably
expressed on the surface of said tumor. In this case, the VH and VL
chains are advantageously coupled to a toxine. The coupling can be
effected on the nucleic acid level by genetic engineering or at the
protein level by, for example, chemical coupling.
It is particularly preferred that said tumor antigen is the 17-1A
antigen.
In a further particularly preferred embodiment of the method of the
invention, said VH chain comprises one of the two sequences shown
in FIG. 7 (nucleotides 1 to 381) and FIG. 8 (nucleotides 1 to 339)
and/or said VL chain comprises one of the two following sequences
shown in FIG. 6 (nucleotides 1 to 321) and FIG. 9 (nucleotides 1 to
321). Receptors with these specific VH and VL regions, wherein said
VL region can be combined with both VH regions, are the first human
antibodies that are specific for the human 17-1A antigen.
In a further preferred embodiment of the method of the invention,
said selection step involves
(i) binding of the display vehicle expressing an antigen
receptor
(a) on immobilized target antigen or fragments thereof; (b) on
optionally labeled cells expressing the target antigen or fragments
thereof; or (c) to soluble, preferably labeled target antigen or
fragments thereof; (ii) washing off non-specifically binding
display vehicle (a and b) and subsequent elution of specifically
binding display vehicle by non-specific (e.g. low pH buffer) or
specific means (e.g. target antigen specific antibody) or (iii)
positive enrichment of target antigen bound display vehicle (b and
c) from target antigen solution or from suspensions of cells
expressing the target antigen for example using magnetic beads
binding to labeled target antigen or labeled cells expressing the
target antigen respectively; thus isolated display vehicles
including their antigen receptors optionally being multiplied by
replication and subjected to further rounds of in vitro selection
as described.
In a further preferred embodiment of the method of the invention,
prior to said selection step either said VH or said VL chain is
selected for binding to said antigen together with a surrogate V
chain.
This two-step procedure can be employed using a target antigen
specific template antibody from a different species, for example a
murine monoclonal antibody against the human target antigen. First,
a human VL- or VH-repertoire is combined with a single surrogate
VH- or VL-chain from the murine template antibody, displayed, e.g.,
on filamentous phage and selected in vitro for antigen binding.
Thus, the complete library size is available exclusively for the
human VL- or VH-repertoire and candidate human VL- or VH-chains can
be isolated that are capable of contributing to specific binding of
the human target antigen. In a second step, the surrogate variable
region of the template antibody is replaced by the corresponding
human variable region repertoire followed by a second round of in
vitro selection; again, the complete library size is exclusively
available for a single VH- or VL-region repertoire, thus enabling
much more VL- and VH-region candidates to be screened for antigen
binding under conditions of limited library size by the two-step
procedure than by a single-step procedure.
For cloning of DNA-sequences encoding the variable regions of human
antibodies that specifically bind to the human 17-1A-antigen, this
two-step selection procedure for screening human IgD-combinatorial
antibody libraries by the phage display method was advantageously
employed. First, the Fd-heavy chain segment (VH+CH1) of the murine
monoclonal antibody M79 (Gottlinger, Int. J. Cancer 38 (1986), 47
53) that specifically binds to the human 17-1A-antigen was combined
with a human kappa- and lambda-light chain repertoire respectively.
The resulting libraries were displayed on filamentous phage and
selected in vitro by several rounds of panning on immobilized
recombinant human 17-1A-antigen. Soluble Fab-fragments were
expressed from several clones after each round of panning and
screened by ELISA for antigen binding. Each of the strong binding
entities enriched during the panning procedure proved to contain
the same human kappa-light chain as confirmed by sequence analysis.
This human light chain furtheron called K8 was then combined with a
human IgD-heavy chain library, that was again displayed on
filamentous phage and selected in vitro by several rounds of
panning on immobilized recombinant human 17-1A-antigen. Several
Fab-fragments were expressed from several clones after each round
of panning and again screened by ELISA for antigen binding.
Sequence analysis of the binding entities enriched during the
panning procedure revealed two different heavy chain-variable
regions called D4.5 and D7.2 each of which combines with the
K8-light chain to form different human antigen binding sites with
specificity for the human 17-1A-antigen. The human light and heavy
chain repertoires were cloned from several preparations of total
RNA isolated from human blood and bone marrow samples of several
donors by using kappa or lambda light chain specific as well as
IgD-heavy chain specific RT-PCR. As it is impossible to selectively
amplify the light chain repertoire that is combined with IgD-heavy
chains in vivo, unless IgD-positive B-cells are purified for
RNA-preparation, the light chain libraries used are not limited to
the antibody repertoire of mature unprimed or anergic
B-lymphocytes. However, due to the predominance of the heavy chain
in antigen recognition, this does not substantially undermine the
advantages of the IgD-repertoire for selecting human antibodies to
self-antigens. Further and most importantly due to the exposure to
the human immune system selection of such light chains still
guarantees a low immunogenic profile in humans.
In a further particularly preferred embodiment of the method of the
invention, said surrogate chain is a mouse VH or VL chain.
In a further preferred embodiment of the method of the invention,
said selection of a suitable combination involves (a) testing one
and the same VH chain in combination with a variety of different VL
chains for binding to said human antigen; or (b) testing one and
the same VL chain in combination with a variety of different VH
chains for binding to said human antigen.
This embodiment is advantageously employed again, if either the VL
or the VH chains are known to specifically interact with the human
target molecule. Then, an appropriate second chain can be selected
on the basis of preferably an improved binding to the target
molecule.
In a further preferred embodiment of the method of the invention,
said method comprises the steps of obtaining, after selection, the
human VH and VL chains or the corresponding nucleic acids and
fusing said chains to the same or other VH or VL chains, to
immunoglobulin constant regions of heavy (CH) or light chains (CL)
or parts thereof or to other biologically active molecules such as
peptides, proteins, nucleic acids, small organic compounds,
hormones, neural transmitters, peptidomimics, PNAs (Milner, Nature
Medicine 1 (1995), 879 880; Hupp, Cell 83 (1995), 237 245; Gibbs
and Oliff, Cell 79 (1994), 193 198). The other functional molecule
may be either physically linked by, e.g., chemical means to VH and
VL chains or may be fused by recombinant DNA techniques well known
in the art.
This embodiment of the invention is particularly useful for
developing specific drugs that may be used to target desired
antigens in the human body. For example, if tumor antigens are
targeted, the VH and VL chains may, at the nucleic acid or amino
acid level, be fused to a toxin moiety, thus resulting in an
immunotoxin, to the extracellular portion of a cellular receptor or
a soluble cytokine or parts thereof respectively, thus resulting in
constructs enhancing the anti-tumor immune response or to an
antibody-Fv-fragment thus resulting in a bispecific antibody
derivative.
In a further particularly preferred embodiment of the method of the
invention, said constant region chains are derived from human IgG1
or IgG3.
The constant region chains of human IgG1 or IgG3 are preferentially
used if cells expressing the target antigen should be destroyed in
the human body. It is well-known in the art that these
IgG-subclasses efficiently mediate antibody dependent cellular
cytotoxicity (ADCC) and contribute to the destruction of cells
recognized and bound by these antibody subclasses.
In a further preferred embodiment of the method of the invention,
said VH and/or VL chains are coupled with non-proteinous
pharmaceuticals preferrably of low molecular weight such as
radioisotopes or substances used for chemotherapy, thus resulting
in a more specific in vivo targeting of said pharmaceuticals.
In a further preferred embodiment of the method of the invention,
said VH or VL chains are expressed from nucleic acid sequences that
are the result of the RT-PCR amplification of mRNA derived from
essentially unprimed mature human B-lymphocytes or essentially
anergic human B-cells.
It is preferred to amplify the VH or VL chains by RT-PCR once the
suitable source thereof has been identified and isolated. It is
preferred to use the mRNA of nucleated cells from human bone marrow
or more preferable from human blood for amplifying VH or VL chains
by RT-PCR as these two tissue compartments are the most easily
accessible B-cell sources in humans. It is further preferred to
isolate anergic B-cells or more preferable mature unprimed
B-lymphocytes from the nucleated cells of said tissue compartments
by using e.g. magnetic beads or flow cytometry based cell sorting
prior to RNA-preparation. This procedure guarantees that both, VH
and VL chains amplified by RT-PCR, are derived from the preferred
B-cell population. Alternatively, if mRNA of the whole fraction of
nucleated cells from said tissue compartments is used to amplify VH
or VL chains by RT-PCR, it is preferable to amplify the VH-region
as half of the heavy chain Fd-segment (VH-CH1) of human IgD by
using an IgD-specific 3' PCR-primer e.g. that enlisted in table 1
which exclusively gives raise to PCR-products from the human
IgD-heavy chain, only expressed in mature unprimed human
B-lymphocytes and in anergic human B-cells.
In a further preferred embodiment of the method of the invention,
the anti-human antigen receptor which is low or not immunogenic in
humans, comprises a combination of functionally rearranged VH and
VL chains preferably from essentially unprimed mature human
B-lymphocytes or essentially anergic human B-cells and obtainable
by the method according to the invention described above.
The advantages of the antibody of the invention have been outlined
herein above. It has to be emphasized that corresponding antibodies
directed against human antigens and derived from human sources,
said antibodies having thus a low or no immunogenicity in humans,
have so far not been isolated in the art. Accordingly, the
antibodies of the invention are the starting point of a whole new
development of antibodies that may be used in various fields of
medicine and pharmacy.
In an additional preferred embodiment of the method of the
invention, the receptor is an antibody or a fragment thereof.
In another preferred embodiment of the method of the invention, the
receptor is specific for a human tumor antigen, most preferably for
the human 17-1A antigen.
The invention furthermore relates to a receptor wherein said VH
chain comprises one of the sequences shown in FIG. 7 (nucleotides 1
to 381) and FIG. 8 (nucleotides 1 to 339) and/or said VL chain
comprises one of the two following sequences shown in FIG. 6
(nucleotides 1 to 321) and FIG. 9 (nucleotides 1 to 321).
Furthermore, the invention relates to a VH chain or a part thereof
comprised in the receptor of the invention.
The invention also relates to a VL chain or a part thereof
comprised in the receptor of the invention.
In a further particularly preferred embodiment of the method of the
invention, said part of said VH chain is the CDR3 domain.
Furthermore, the invention relates to a kit comprising a
combination of functionally rearranged VH and VL immunoglobulin
chains wherein at least one of the VH and VL chains are derived
from essentially unprimed mature human B lymphocytes or from
essentially anergic human B cells, said chains being expressible
from recombinant vectors of an in vitro display system.
Said kit is advantageously used in carrying out the method of the
invention and thus obtaining receptors of desired specificity.
Preferably, in said kit, said in vitro display system is a phage
display system.
The invention relates further to an antibody characterized in that
it is derived from human sequences and is specific for the human
17-1A antigen.
With the method of the invention, for the first time a human
antibody which is specific for the human 17-1A antigen has been
developed. As has been pointed out before, this development was no
trivial task, since human antibodies against human tumor associated
antigens are usually subjected to mechanisms mediating self
tolerance of the immune system, thus resulting in the elimination
of B-lymphocytes expressing autoreactive antibodies in vivo. Among
the known tumor associated antigens 17-1A is by far more
ubiquitously expressed on a broad range of normal epithelial
tissues than other tumor antigens, in addition to its expression on
epithelial tumors.
Therefore, the 17-1A-antigen is currently regarded to represent a
pan-epithelial antigen rather than a tumor antigen, which it was
thought to be at the time of its first description. In comparison,
other so called tumor antigens found on epithelial tumors, like
erb-B2 (Her 2/neu), Muc-1 (PEM) or the Thompson-Friedreich-antigen
usually show a much more restricted expression pattern on normal
epithelial tissues. Thus, the selective force of self tolerance
against B-lymphocytes expressing 17-1A reactive antibodies even
with low affinity is expected to be exceptionally high, since it
appears nearly impossible for such B-cells to avoid encounters with
the 17-1A antigen for longer periods of time, due to the ubiquity
of this antigen. Therefore, it was surprisingly found in accordance
with the present invention, that 17-1A specific antibodies or at
least the corresponding heavy chains can be isolated from the
antibody repertoire of human B-cells such as e.g. mature unprimed
B-lymphocytes. These B-cells are known to have a long in vivo half
life and have already managed to avoid depletion prior to their
maturation despite the presence of the 17-1A antigen even at the
site of maturation; B-cell anergy does not occur in case of the
17-1A antigen as this type of B-cell tolerance is only induced by
soluble self antigen. Only long-lived B-cells that managed to
survive in spite of their potential 17-1A reactivity represent a
repertoire of human immunoglobulin variable regions, that is likely
to be well tolerated by the human immune system when used for the
construction of human antibodies and antibody derivatives designed
to be repeatedly administered systemically to human beings since it
has already been screened for and subsequently eliminated from
immunogenic sequences by the surveillance function of the immune
system. Therefore, the present invention also relates to human
antibodies specific for the 17-1A antigen and suited for repeated
in vivo application regardless of the method by which they are
obtained as human antibody sequences with said high in vivo
compatibility are highly expected to be also found in the B-cell
repertoire of healthy human beings, for example, by the method of
the invention. Accordingly, for the first time now a human antibody
has been developed that can advantageously be used in the
monitoring and/or destruction of tumor cells carrying the 17-1A
antigen.
Thus, the antibody of the invention is advantageously low or
non-immunogenic in humans. In a preferred embodiment, said antibody
which is obtainable according to a method as described hereinabove.
In another preferred embodiment, the antibody of the invention
recognizes an epitope of the extracellulary domain of the 17-1A
antigen preferably comprising at least one amino acid sequence of
peptide Nos. 8, 11, 13, 14, 59, 60, 77 and 79. Preferably, the VH
chain of the antibody of the invention comprises at least one CDR
of one of the following two sequences shown in FIG. 7 (nucleotides
1 to 381) and FIG. 8 (nucleotides 1 to 339) and/or the VL chain
comprises at least one CDR of the following two sequences shown in
FIG. 6 (nucleotides 1 to 321) and FIG. 9 (nucleotides 1 to 321).
Particularly preferred is an antibody, wherein said VH chain
comprises one of the two following sequences shown in FIG. 7
(nucleotides 1 to 381) and FIG. 8 (nucleotides 1 to 339) and/or
said VL chain comprises one of the following two sequences shown in
FIG. 6 (nucleotides 1 to 321) and FIG. 9 (nucleotides 1 to
321).
This receptor and preferably antibody repertoire selected for low
immunogenicity has been concluded to be best represented in a human
IgD-antibody library. IgD is expressed as membrane antigen receptor
together with surface IgM on mature unprimed B-lymphocytes that
enter primary follicles during their traffic to and between
secondary lymphoid organs unless they have encountered multivalent
self antigen resulting in clonal deletion or soluble monovalent
self antigen rendering them anergic and short lived due to
exclusion form primary follicles. Except mature unprimed
B-lymphocytes human IgD-libraries only represent the antibody
repertoire of short-lived B-cells that have been rendered anergic
in contact with soluble monovalent self antigen but are unlikely to
contribute specific binding entities to human cell surface
molecules resembling multivalent self-antigens that induce clonal
deletion instead of B-cell anergy.
Moreover, the present invention relates to a pharmaceutical
composition comprising at least one of the aforementioned receptors
or parts thereof of the invention, either alone or in combination,
and optionally a pharmaceutically acceptable carrier or excipient.
Examples of suitable pharmaceutical carriers are well known in the
art and include phosphate buffered saline solutions, water,
emulsions, such as oil/water emulsions, various types of wetting
agents, sterile solutions etc. Compositions comprising such
carriers can be formulated by well known conventional methods.
These pharmaceutical compositions can be administered to the
subject at a suitable dose. Administration of the suitable
compositions may be effected by different ways, e.g. by
intravenous, intraperitoneal, subcutaneous, intramuscular, topical
or intradermal administration.
Thus, the invention also relates to the use of a receptor or parts
thereof produced according to the method of the invention for the
preparation of a pharmaceutical composition for treating,
preventing and/or delaying of a tumor, in a subject, preferably
wherein the tumor is of epithelial origin.
The figures show:
FIG. 1: Cloning site of pComb3H with important restriction sites.
The following abbreviations were used: P, promotor; VL, variable
light chain domain; CL, constant light chain domain; VH, variable
heavy chain domain; CH1, constant heavy chain domain 1; L1/2,
procaryotic leader sequences. The domain designated as gene III in
pComb3H encodes for the non-infectious part of the gene III-product
of filamentous phages as e.g. VCSM13.
FIG. 2: Scheme of the pComb3H-plasmid and the fully expressed
M13-phage. On pComb3H the organization of leader (L) ompA, light
chain, leader (L) pelB, heavy chain and gene III is shown. The
fully expressed M13-phage displays on its surface the phenotype of
a certain Fab antibody-fragment consisting of a light chain and the
Fd-segment (VH+CH1) of a heavy chain linked to the non-infectious
part of the gene III product and contains the corresponding
genotype as single-stranded DNA encoding the heavy and light chain
of the displayed Fab-fragment. The infectious gene III-protein is
provided by the helper phage VCSM13.
FIG. 3: ELISA of soluble Fab fragments. Periplasma preparations of
soluble Fab fragments each containing the Fd segment of chimerized
M79 and a single human kappa chain per clone. ELISA plates were
incubated overnight with soluble 17-1A-antigen. Detection of bound
Fab-antibody fragments was carried out with a peroxidase-conjugated
polyclonal anti-human immunoglobulin antibody. The ELISA was
finally developed by adding an ABTS-substrate solution (ABTS=2,2
Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid)) and the substrate
turnover measured at a wavelength of 405 nm (y-axis). Clones are
presented on the x-axis, the first number indicates the round of
panning, the second one is the clone number. Clones 1.5 9 have a
combination of chimeric M79 Fd segment with one random kappa chain
and represent negative controls.
FIG. 4: ELISA of soluble Fab fragments. Periplasma preparations of
soluble Fab fragments each containing the k8 light chain and a
single human Ig delta chain Fd-segment. ELISA plates were incubated
overnight with soluble 17-1A-antigen. Detection of bound
Fab-antibody fragments was carried out with a biotinylated
polyclonal anti-human kappa light chain antibody followed by
peroxidase-conjugated streptavidine. The ELISA was finally
developed by adding an ABTS-substrate solution (ABTS=2,2
Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid)) and the substrate
turnover measured at a wavelength of 405 nm (y-axis). Clones are
presented on the x-axis, the first number indicates the round of
panning, the second one is the clone number. Clones 1.2 6 have a
combination of k8 light chain with one random Ig delta heavy chain
Fd-segment and represent negative controls.
FIG. 5: ELISA of soluble Fab fragments. Periplasma preparations of
soluble Fab fragments each containing the D4.5 Fd segment and a
single human kappa chain per clone. ELISA plates were incubated
overnight with soluble 17-1A-antigen. Detection of bound
Fab-antibody fragments was carried out with a biotinylated
polyclonal anti-human kappa light chain antibody followed by
peroxidase-conjugated streptavidine. The ELISA was finally
developed by adding an ABTS-substrate solution (ABTS=2,2
Azino-bis(3-Ethylbenzthiazoline-6-Sulfonic Acid)) and the substrate
turnover measured at a wavelength of 405 nm (y-axis). Clones are
presented on the x-axis, the first number indicates the round of
panning, the second one is the clone number; S. is the abbreviation
for clones prior to the first round of selection. Clones S.1 3 have
a combination of k8 light chain with one random Ig delta heavy
chain Fd-segment and represent negative controls.
FIG. 6: DNA- and protein-sequence of the human kappa 8 light chain
variable region. Numbers indicate the nucleotide (nt) positions,
amino acids are presented in single letter code. CDR1 includes nt
70 to nt 102, CDR2 nt 148 to nt 168, CDR3 nt 265 to nt 294 (SEQ ID
NO:141).
FIG. 7: DNA-sequence of the human D4.5 heavy chain variable region.
Numbers indicate the nucleotide (nt) positions, amino acids are
presented in the single letter code. CDR1 includes nt 91 to nt 105,
CDR2 nt 148 to nt 198, CDR3 nt 292 to nt 351. The border between
the heavy chain variable region and the CH1 domain of the Ig delta
constant region is located between nt 382 an 383 with the delta
constant region protein sequence starting at nt 384 (SEQ ID
NO:143).
FIG. 8: DNA-sequence of the human D7.2 heavy chain variable region.
Numbers indicate the nucleotide (nt) positions, amino acids are
presented in the single letter code. CDR1 includes nt 91 to nt 105,
CDR2 nt 148 to nt 198, CDR3 nt 292 to nt 309. The border between
the heavy chain variable region and the CH1 domain of the Ig delta
constant region is located between nt 340 and nt 31 with the delta
constant region protein sequence starting at nt 343 (SEQ ID
NO:145).
FIG. 9: DNA- and protein-sequence of the human kappa 5.1 light
chain variable region. Numbers indicate the nucleotide (nt)
positions, amino acids are presented in single letter code. CDR1
includes nt 70 to nt 102, CDR2 nt 148 to nt 168, CDR3 nt 265 to 294
(SEQ ID NO:147).
FIG. 10: Flow cytometry analysis of Fab antibody-fragments and
control antibody M79 on 17-1A positive Kato cells for testing
binding activity of a periplasma preparation containing the
k8-D4.5-Fab fragment. Kato-cells were incubated with I) irrelevant
periplasma preparation, II) 10 .mu.g/ml chimeric (bivalent !) M79,
III) k8-D4.5 periplasma preparation and IV) 1:10 dilution of
K8-D4.5 periplasma preparation. Relative cell numbers are shown on
the y-axis, relative fluorescense intensity is shown on the
x-axis.
FIG. 11: Flow cytometry analysis of Fab antibody-fragments and
complete antibodies on 17-1A positive Kato cells, 17-1A transfected
and untransfected CHO-cells, respectively, for testing binding
activity of periplasma preparations (pp) containing the k8-D4.5-,
the k5.1-D4.5- or an irrelevant k-D4.5-Fab-fragment. Kato-cells
were incubated I) with irrelevant pp (broken line) or k5.1-D4.5-pp
(solid line), or II) with 20 .mu.g/ml M79-antibody (solid line) or
the corresponding murine IgG2a isotype control (broken line).
17-1A-transfected CHO-cells were incubated III) with irrelevant pp
(broken line) or k5.1-D4.5-pp (solid line), IV) with irrelevant pp
(broken line) or k8-D4.5-pp (solid line), or V) with 20 .mu.g/ml
M79-antibody (solid line) or the corresponding murine IgG2a isotype
control (broken line). In III), IV) and V) incubation and detection
of relevant pp (k5.1-D4.5Fab-pp and k8-D4.5-Fab-pp, respectively)
and the murine antibody M79 was also carried out on untransfected
CHO-cells (dotted lines). Relative cell numbers are shown on the
y-axis, relative fluorescense intensity is shown on the x-axis.
FIG. 12: Cloning site of pEF-ADA with important restriction sites.
The following abbreviations were used: P, promotor; VL, variable
light chain domain; CL, constant light chain; Leuc, eucaryotic
leader sequence.
FIG. 13: Cloning site of pEF-DHFR with important restriction sites.
The following abbreviations were used: P, promotor; VH, variable
heavy chain domain; CH1/2/3, constant heavy chain domain 1/2/3;
Leuc, eucaryotic leader sequence.
FIG. 14: SDS-PAGE of preparations of the human antibody H79.
Approx. 10 .mu.g of each antibody were run on a 12.5% denaturating
Polyacrylamid-gel under reducing and non-reducing conditions and
stained with coomassie blue. Lane 1: Marker (MW [kDa] of single
bands marked on the left side of the gel) Lane 2: H79 human
IgG1-version (non-reducing) Lane 3: H79 human IgG1-version under
reducing conditions Lane 4: H79 murine IgG1-version (non-reducing)
Lane 5: H79 murine IgG1-version under reducing conditions
FIG. 15: SDS-PAGE of preparations of the human antibodies H79 and
HD70. 10 .mu.g of H79 and 3.5 .mu.g HD70 were run on 12.5%
denaturating Polyacrylamid-gel under non-reducing (gel 1) and
reducing (gel 2) conditions and stained with coomassie blue. Gel 1:
Lane 1: Marker (MW [kDa] of single bands marked on the left side of
the gel) Lane 2: H79 human IgG1-version (non-reducing) Lane 3: HD70
human IgG1-version (non-reducing) Gel 2: Lane 4: Marker (MW [kDa]
of single bands marked on the left side of the gel) Lane 5H79 human
IgG1-version under reducing conditions Lane 6 HD70 human
IgG1-version under reducing conditions
FIG. 16: Flow cytometry analysis of 17-1A positive Kato cells for
testing binding activity of purified H79 human IgG1 as well as
purified H79 murine IgG1. Kato-cells were incubated with IgG
isotype-controls (10 .mu.g/ml human IgG1 and murine IgG1,
respectively), positive controls (M79 and chimerized M74 at 10
.mu.g/ml, respectively; (both 17-1A specific) and with H79 human
IgG1 or H79 murine IgG1 (10 .mu.g/ml and 1 .mu.g/ml each). Relative
cell numbers are shown on the y-axis, relative fluorescense
intensity is shown on the x-axis.
FIG. 17: Flow cytometry analysis of antibodies (conzentration: 20
.mu.g/ml each) on 17-1A positive Kato cells, 17-1A-transfected and
non-transfected CHO-cells for testing binding activity of the
purified antibodies H79 human IgG1, H79 murine IgG1, HD70, D7.2,
M79, Panorex and isotype controls (human IgG1, murine IgG1 and 2a).
I) H79 human IgG1 (solid line) and human IgG1 isotype control
(broken line) on Kato-cells, II) H79 murine IgG1 (solid line) and
murine IgG1 isotype control (broken line) on Kato-cells, III) HD70
(solid line) and human IgG1 isotype control (broken line) on
Kato-cells, IV) D7.2 (solid line) and human IgG1 isotype control
(broken line) on Kato-cells, V) M79 (solid line) and murine IgG2a
isotype control (broken line) on Kato-cells, VI) Panorex (solid
line) and murine IgG2a isotype control (broken line) on Kato-cells,
VII) H79 human IgG1 (solid line) and human IgG1 isotype control
(broken line) on 17-1A-transfected CHO-cells, VIII) H79 murine IgG1
(solid line) and murine IgG1 isotype control (broken line) on
17-1A-transfected CHO-cells, IX) HD70 (solid line) and human IgG1
isotype control (broken line) on 17-1A-transfected CHO-cells, X)
D7.2 (solid line) and human IgG1 isotype control (broken line) on
17-1A-transfected CHO-cells, XI) M79 (solid line) and murine IgG2a
isotype control (broken line) on 17-1A-transfected CHO-cells, XII)
Panorex (solid line) and murine IgG2a isotype control (broken line)
on 17-1A-transfected CHO-cells. Figures VII XII also show the
results of incubation and detection of the relevant antibodies on
non-transfected CHO-cells (dotted line).
FIG. 18: .sup.51Cr antibody dependent cellular cytotoxicity assay.
For .sup.51Cr release unstimulated human peripheral blood
mononuclear cells (PBMCs, 5.times.10.sup.5 cells) as effector cells
were incubated with labelled target cells (Kato-cells labelled for
2 h with .sup.51Cr) and antibodies in different concentrations for
4 or 20 h at 37.degree. C. Corresponding non-binding isotypes
(h-IgG=human IgG1; m-IgG2a=murine IgG2a) were used as negative
controls (H79=H79huIgG1). Specific lysis was calculated as ((cpm,
experimental release)-(cpm, spontaneous release))/((cpm, maximal
release)-(cpm, spontaneous release)).
FIG. 19: Light microscopic photo of healthy human colon tissue
stained with M79 (positive) control. 5 nm cryosections of normal
mucosa tissue were incubated with the murine M79 antibody (IgG2a)
as positive control (10 .mu.g/ml). Detection of bound murine
antibodies was carried out with a peroxidase conjugated polyclonal
anti-mouse-Ig antibody and stained with carbazole (brown). Counter
staining was carried out with hemalaun (blue).
FIG. 20: Light microscopic photo of healthy human colon tissue
stained with H79 murine IgG1. 5 nm cryosections of normal mucosa
tissue were incubated with the murine IgG version of the H79
antibody (10 .mu.g/ml). Detection of bound murine IgG1 antibodies
was carried out with a peroxidase conjugated polyclonal
anti-mouse-Ig antibody and stained with carbazole (brown). Counter
staining was carried out with hemalaun (blue).
FIG. 21: Light microscopic photo of colon carcinoma stained with
M79 (positive control). 5 nm cryosections colon carcinoma were
incubated with the murine M79 antibody (IgG2a) as positive control
(10 .mu.g/ml). Detection of bound murine antibodies was carried out
with a peroxidase conjugated polyclonal anti-mouse Ig antibody and
stained with carbazole (brown). Counter staining was carried out
with hemalaun (blue).
FIG. 22: Light microscopic photo of colon carcinoma stained with
H79 murine IgG1. 5 nm cryosections of colon carcinoma tissue were
incubated with the murine IgG1 version of the H79 antibody (10
.mu.g/ml). Detection of bound murine IgG1 antibodies was carried
out with a peroxidase conjugated polyclonal anti-mouse-Ig antibody
and stained with carbazole (brown). Counter staining was carried
out with hemalaun (blue).
FIG. 23: Light microscopic photo of healthy human colon tissue
stained with murine IgG2a isotype control. 5 nm cryosections of
normal mucosa tissue were incubated with irrelevante murine IgG2a
antibody as negative control (10 .mu.g/ml). Detection of bound
murine antibodies was carried out with a peroxidase conjugated
polyclonal anti-mouse-Ig antibody and stained with carbazole
(brown). Counter staining was carried out with hemalaun (blue).
FIG. 24: Light microscopic photo of healthy human colon tissue
stained with IgG1 isotype control. 5 nm cryosections of normal
mucosa tissue were incubated with irrelevante murine IgG1 antibody
as negative control (10 .mu.g/ml). Detection of bound murine
antibodies was carried out with a peroxidase conjugated polyclonal
anti-mouse-Ig antibody and stained with carbazole (brown). Counter
staining was carried out with hemalaun (blue).
FIG. 25: Light microscopic photo of human colon carcinoma tissue
stained with murine IgG2a isotype control. 5 nm cryosections of
normal mucosa tissue were incubated with irrelevante murine IgG2a
antibody as negative control (10 .mu.g/ml). Detection of bound
murine antibodies was carried out with a peroxidase conjugated
polyclonal anti-mouse-Ig antibody and stained with carbazole
(brown). Counter staining was carried out with hemalaun (blue).
FIG. 26: Light microscopic photo of human colon carcinoma tissue
stained with IgG1 isotype control. 5 nm cryosections of normal
mucosa tissue were incubated with irrelevante murine IgG1 antibody
as negative control (10 .mu.g/ml). Detection of bound murine
antibodies was carried out with a peroxidase conjugated polyclonal
anti-mouse-Ig antibody and stained with carbazole (brown). Counter
staining was carried out with hemalaun (blue).
FIG. 27: Epitope analysis of murine antibody M79 and human
antibodies H79 and HD70, each of which is specific for the human
17-1A antigen. Antibodies were incubated with a gridded array of
peptides comprising 119 polypeptides of 13 amino acids, shifted by
two amino acids and covering the entire extracellular amino acid
sequence of the human 17-1A-antigen. The peptides were covalently
attached at their C-termini to a Pep Spots Membrane (Jerini
Biotools, Berlin) as individual spots. Bound murine M79 antibody
was directly detected on the Pep Spots Membrane by an anti-murine
immunoglobulin antibody coupled to horse radish peroxidase (HRP)
followed by chemoluminescence. Signals that are due to the
reactivity of the secondary antibody alone with single peptide
spots are shown in the corresponding control staining of the Pep
Spots Membrane. By subtracting the main background staining at
peptide spot 87, peptide spots 38 and 95 turn out to represent
specific binding of murine antibody M79. Due to a higher degree of
crossreactivity of the secondary HRP-conjugated anti-human
immunoglobulin antibody with several peptide spots, bound human
antibodies H79 and HD70 were transfered from the Pep Spots Membrane
to a blotting membrane, respectively, by means of electrotransfer
and subsequently detected by said anti human secondary antibody and
chemoluminescence. Specific binding of human antibody H79 could be
detected mainly at peptide spots 8, 11, 13, 14, 59 60, 77 and 79;
however, no binding was detectable in case of human antibody
HD70.
The Examples illustrate the invention:
EXAMPLE I
Construction of the Combinatorial Antibody Libraries and Phage
Display
A library of human immunoglobuline (Ig) light chain and Ig heavy
chain Fd-DNA-fragments was constructed by RT-PCR with kappa-,
lambda- and Fd delta specific primer sets on the total RNA prepared
from peripheral blood lymphocytes (PBL)- and bone marrow-samples of
four and ten human donors, respectively according to Chomczynski,
Analytical Biochemistry 162 (1987) 156 159. cDNA was synthesized
according to standard methods (Sambrook, Cold Spring Harbor
Laboratory Press 1989, second edition).
The following primer sets were chosen, giving rise to a 5'-XhoI and
a 3'-SpeI recognition site for the heavy chain fragments and a
5'-SacI and a 3'-XbaI recognition site for light chains:
For the PCR-amplification of the delta Fd cDNA-fragments five
different 5'-VH-family specific primers were each combined with one
3'-CH1 delta primer; for the PCR-amplification of the kappa (K)
light chain fragments five different 5'-VK-family specific primers
were each combined with one 3'-CK primer and for the amplification
of the lambda (L) light chain fragments, eight different
5'-VL-family specific primers were combined with one
3'-CL-primer.
Primer sets for the amplification of the Fab DNA-fragments (5' to
3') are shown in Table I below.
The following PCR-program was used for amplification:
Denaturation at 94.degree. C. for 15 seconds, primer annealing at
52.degree. C. for 50 seconds and primer extension at 72.degree. C.
for 90 seconds for 40 cycles, followed by a 10 minutes final
extension at 72.degree. C.
TABLE-US-00001 TABLE I Primer sets heavy chain Fd-fragment:
5'-primer: VH1,3,5,7: AGGTGCAGCTGCTCGAGTCTGG SEQ ID NO.:1 VH2:
CAG(AG)TCACCTTGCTCGAGTCTGG SEQ ID NO.:2 VH4:
CAGGTGCAGCTGCTCGAGTCGGG SEQ ID NO.:3 VH4B: CAGGTGCAGCTACTCGAGTGGGG
SEQ ID NO.:4 VH6: CAGGTACAGCTGCTCGAGTCAGG SEQ ID NO.:5 3'-primer:
CD1: TGCCTTACTAGTCTCTGGCCAGCGGAAGAT SEQ ID NO.:6 kappa chain
fragment: 5'-primer: VK1: GAGCCGCACGAGCCCGAGCTCCAGATGACCCAGTCTCC
SEQ ID NO.:7 VK3: GAGCCGCACGAGCCCGAGCTCGTG(AT)TGAC(AG)CAGTCTCC SEQ
ID NO.:8 VK2/4: GAGCCGCACGAGCCCGAGCTCGTGATGAC(CT)CAGTCTCC SEQ ID
NO.:9 VK5: GAGCCGCACGAGCCCGAGCTCACACTCACGCAGTCTCC SEQ ID NO.:10
VK6: GAGCCGCACGAGCCCGAGCTCGTGCTGACTCAGTCTCC SEQ ID NO.:11
3'-primer: CK1D: GCGCCGTCTAGAATTAACACTCTCCCCTGTTGAAGCTCTTTGTGA SEQ
ID NO.:12 CGGGCGAACTCAG lambda chain fragment: 5'-primer: VL1:
AATTTTGAGCTCACTCAGCCCCAC SEQ ID NO.:13 VL2:
TCTCCGAGCTCCAGCCTGCCTCCGTG SEQ ID NO.:14 VL3:
TCTGTGGAGCTCCAGCCGCCCTCAGTG SEQ ID NO.:15 VL4:
TCTGAAGAGCTCCAGGACCCTGTTGTGTCTGTG SEQ ID NO.:16 VL5:
CAGTCTGAGCTCACGCAGCCGCCC SEQ ID NO.:17 VL6:
CAGACTGAGCTCACTCAGGAGCCC SEQ ID NO.:18 VL7:
CAGGTTGAGCTCACTCAACCGCCC SEQ ID NO.:19 VL8:
CAGGCTGAGCTCACTCAGCCGTCTTCC SEQ ID NO.:20 3'-primer: CL2:
CGCCGTCTAGAATTATGAACATTCTGTAGG SEQ ID NO.:21
450 ng of the kappa light chain fragments (digested with SacI and
XbaI) were ligated with 1400 ng of the phagmid pComb3H (digested
with SacI and XbaI; large DNA-fragment) derived from pComb3
(Barbas, Proc. Natl. Acad. Sci U.S.A. 88 (1991) 7978 7982) wherein
the heavy chain position was already occupied by the Fd fragment of
the chimerized murine antibody M79 (containing a human IgG1 CH1)
directed against the extracellular part of the 17-1A protein (see
FIG. 1 for pComb3H cloning site).
The resulting combinatorial antibody DNA library was then
transformed into 300 .mu.l of electrocompetent Escherichia coli XL1
Blue by electroporation (2.5 kV, 0.2 cm gap cuvette, 25 FD, 200
Ohm, Biorad gene-pulser) thus resulting in a library size of
4.times.10.sup.7 independent clones. After one hour of phenotype
expression, positive transformants were selected for carbenicilline
resistance encoded by the pComb vector. After this adaption these
clones were infected with an infectious dose of 1.times.10.sup.12
phage particles of the helper phage VCSM13 resulting in the
production and secretion of filamentous M13 phages, each of them
containing single stranded pComb3H-DNA encoding a single human
light chain and the Fd segment of chimeric M79 and displaying the
corresponding Fab fragment on the phage surface as a translational
fusion to the non-infectious part of phage coat protein III (phage
display), see FIG. 2.
This phage library carrying the cloned Fab repertoire was harvested
from the culture supernatant by PEG8000/NaCl precipitation and
centrifugation, redissolved in TBS/1% BSA and incubated with
recombinant s17-1A immobilized on 96 well ELISA plates. s17-1A was
prepared as described (Mack, Proc. Natl. Sci. U.S.A. 92 (1995) 7021
7025). Fab phages that did not specifically bind to the target
antigen were eliminated by up to ten washing steps with TBS/0.5%
Tween. Binders were eluted by using HCl-Glycine pH 2.2 and after
neutralization of the eluat with 2 M Tris pH 12, used for infection
of a new uninfected E. coli XL1 Blue culture. Cells successfully
transduced with a pComb phagmid copy, encoding an antigen binding
Fab fragment, were again selected for carbenicilline resistance and
subsequently infected with VCSM13 helper phage to start the second
round of antibody display and in vitro selection.
After five rounds of production and selection of antigen-binding
Fab phages, plasmid DNA containing the selected Fab repertoire was
prepared.
For the production of soluble Fab proteins the gene III DNA
fragment was excised from the plasmids thus destroying the
translational fusion of the Fd heavy chain segment with the gene
III protein. After religation, this pool of plasmid DNA was
transformed into 100 .mu.l heat shock competent E. coli XL1 Blue
and plated on Carbenicilline (Carb) LB-Agar. Single colonies were
grown in 10 ml LB-Carb-cultures/20 mM MgCl.sub.2 and Fab expression
was induced after six hours by adding
Isopropyl-.beta.-D-thiogalactosid (IPTG) to a final concentration
of 1 mM. This in vitro selection as well as expression of soluble
Fab-fragments was carried out according to Burton, Proc. Natl.
Acad. Sci. U.S.A. 88 (1991), 10134 10137. The cells were harvested
after 20 hours; periplasma preparation was carried out by four
rounds of freezing (ethanol/dry ice) and thawing (37.degree. C.)
and tested by ELISA for Fab fragments binding to s17-1A. 23 of 27
clones showed binding activity. After sequenzing, the two clones
with the strongest signals (see FIG. 3) turned out to have
identical kappa chains and were called k8; see FIG. 6.
This human kappa light chain k8 was now used as a binding partner
for the human Ig delta heavy chain pool; 2250 ng of human delta
heavy chain Fd DNA-fragments (digested with XhoI and SpeI) were
ligated with 7000 ng of the phagmid vector pComb3H (digested with
XhoI and SpeI; large DNA-fragment) containing the k8 DNA-fragment
in the light chain position.
The choice of the human delta chain repertoire as source for heavy
chain variable-regions that specifically bind to the 17-1A antigen,
when combined with the k8 light chain, appeared to be most
suitable. Delta chains are only produced in mature unprimed and in
self-antigen specific anergic B-cells that have not yet or will not
undergo proliferation; therefore the diversity of their heavy chain
repertoire is higher and the number of each single specificity
therein is lower compared to heavy chain repertoires of other
immunoglobulin isotypes.
The transformation of the pComb-k8-delta Fd-fragment library into a
total of 1500 .mu.l E. coli XL1 Blue by five equal electroporations
(2.5 kV, 0.2 cm gap cuvette, 25 FD, 200 Ohm) resulted in a final
number of 1.1.times.10.sup.9 independent clones. In vitro selection
of this combinatorial antibody library was carried out as described
above for the human light chain repertoire. After four rounds of
panning soluble Fab fragments were prepared from eight clones.
The periplasma preparations were tested on ELISA. One of the clones
showed strong antigen binding (see FIG. 4). This clone was called
D4.5 and the DNA of the Fd delta fragment was sequenzed with a
reverse delta CH1-specific primer (see FIG. 7).
Another s17-1A binding Fab fragment was isolated after further
rounds of panning first appearing in round seven with a markedly
weaker ELISA signal compared to D4.5 (see FIG. 4). The clone was
designated as D7.2 and the DNA sequence was determined again using
the delta specific primer (see FIG. 8).
To identify further light chain partners that combine with the D4.5
delta heavy chain Fd segment to form a 17-1A specific Fab-fragment,
a reshuffling experiment was carried out:
450 ng of human kappa light chain fragments and 450 ng of human
lambda light chain fragments (both digested with SacI and XbaI)
were each ligated with 1400 ng of the phagmid pComb3H (digested
with SacI and XbaI; large DNA-fragment) wherein the heavy chain
position was already occupied by the D4.5 Fd fragment. The
resulting combinatorial antibody libraries (kappa and lambda) were
then each transformed into 300 .mu.l of electrocompetent
Escherichia coli XL1 Blue by electroporation (2.5 kV, 0.2 cm gap
cuvette, 25 FD, 200 Ohm, Biorad gene-pulser) thus resulting in a
library size of 0.5.times.10.sup.7 independent clones for the kappa
library and of 1.4.times.10.sup.7 independent clones for the lambda
library. After one hour of phenotype expression, positive
transformants were selected for carbenicilline resistance encoded
by the pComb vector. After this adaption these clones were infected
with an infectious dose of 1.times.10.sup.12 phage particles of the
helper phage VCSM13 resulting in the production and secretion of
filamentous M13 phages, each of them containing single stranded
pComb3H-DNA encoding a single human light chain and the D4.5 heavy
chain Fd segment and displaying the corresponding Fab fragment on
the phage surface as a translational fusion to the non-infectious
part of phage coat protein III.
Both phage libraries carrying the cloned Fab repertoires (kappa and
lambda) were harvested from the culture supernatant by PEG8000/NaCl
precipitation and centrifugation, redissolved in TBS/1% BSA and
incubated with recombinant s17-1A immobilized on 96 well ELISA
plates. Fab phages that did not specifically bind to the target
antigen were eliminated by up to ten washing steps with TBS/0.5%
Tween. Binders of both libraries (kappa and lambda) were eluted by
using HCl-Glycine pH 2.2 and after neutralization of the eluat with
2 M Tris pH 12, used for infection of new uninfected E. coli XL1
Blue cultures, one for the kappa and one for the lambda library.
Cells successfully transduced with a pComb phagmid copy, encoding
an antigen binding Fab fragment, were again selected for
carbenicilline resistance and subsequently infected with VCSM13
helper phage to start the second round of antibody display and in
vitro selection.
After five rounds of production and selection of antigen-binding
Fab phages, preparations of plasmid DNA were carried out containing
the selected Fab repertoire of each round of panning,
respectively.
For the production of soluble Fab proteins the gene III DNA
fragment was excised from the plasmids thus destroying the
translational fusion of the Fd heavy chain segment with the gene
III protein. After religation, this pool of plasmid DNA was
transformed into 100 .mu.l heat shock competent E. coli XL1 Blue
and plated on Carbenicilline (Carb) LB-Agar. Single colonies were
grown in 10 ml LB-Carb-cultures/20 mM MgCl.sub.2 and Fab expression
was induced after six hours by adding
Isopropyl-.beta.-D-thiogalactosid (IPTG) to a final concentration
of 1 mM. The cells were harvested after 20 hours; periplasma
preparation was carried out by freezing and thawing and tested by
ELISA for Fab fragments binding to s17-1A.
In total, 45 clones of the kappa and 45 clones of the lambda
library derived predominantly from the fifth round of panning but
to a minor extend also from other rounds were tested for binding to
the 17-1A antigen. Only one clone designated k5.1 (kappa library)
that appeared in round Five showed binding activity (see FIG. 5).
The DNA-sequence of the Kappa V-region of k5.1 was determined (see
FIG. 9).
EXAMPLE II
Bacterial Expression in E. coli XL1 Blue
As previously mentioned in example 1, E. coli XL1 Blue transformed
with pComb3H containing one light and the Fd-segment of one heavy
chain produce soluble Fab in sufficient amounts after excision of
the gene III fragment and induction with IPTG. The heavy chain
Fd-segment and the light chain are exported into the periplasma
where they assemble and form functional Fab-fragments.
For better periplasma preparations the cells were grown in
SB-medium supplemented with 20 mM MgCl.sub.2 and are redissolved in
PBS after harvesting. By four rounds of freezing at -70.degree. C.
and thawing at 37.degree. C., the outer membrane of the bacteria
was destroyed by temperature shock and the soluble periplasmatic
proteins including the Fab fragments were released into the
supernatant. After elimination of intact cells and cell-debris by
centrifugation, the supernatants containing the
Fab-antibody-fragments were collected and used for further
examination.
First, k8-D4.5-Fab and k5.1-D4.5-Fab periplasma preparations were
tested for binding to immobilized s17-1A antigen, both showing
strong ELISA signals (see example 1).
Detection of k8-D4.5 and k5.1-D4.5-Fab-fragments bound to
immobilized s17-1A anigen was carried out using a polyclonal
biotinylated anti-human-kappa light chain antibody (1 .mu.g/ml PBS)
detected with horse raddish conjugated Avidine (1 .mu.g/ml PBS).
The signal was developed by adding a substrate solution, containing
2,2'Azino-bis(3-Ethylbenz-Thiazoline-6-Sulfonic Acid) and
Na-perborate and detected at a wavelength of 405 nm.
The test for binding of the two 17-1A positive human Fab-fragments
(k8-D4.5-Fab and k5.1-D4.5-Fab) on Kato-cells (17-1A expressing
gastric cancer cell-line), 17-1A transfected CHO-cells (CHO/17-1A)
and non-transfected CHO-cells was again carried out with the
periplasma preparations. CHO transfected cell-lines were generated
by subcloning of a DNA-fragment encoding the complete amino acid
sequence of the 17-1A-antigen also known as GA733-2 (Szala, Proc.
Natl. Acad. Sci. U.S.A. 87 (1990), 3542 3546), into the eucaryotic
expression vector pEF-DHFR (Mack, Proc. Natl. Acad. Sci. U.S.A. 92
(1995) 7021 7025) according to standard procedures (Sambrook,
Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring
Harbour Laboratory Press, Cold Spring Harbour, N.Y. (1989)). The
resulting plasmid was linearized with NdeI and transfected into
DHFR-deficient CHO-cells for stable expression. The expression of
transmembrane 17-1A was increased by stepwise gene amplification
induced by subsequent addition of increasing concentrations of the
DHFR-inhibitor Methotrexat (MTX) to a final concentration of 500
nM, with the concentration steps in between being 20 nM and 100 nM
(Kaufmann, Methods Enzymol. 185 (1990), 537 566).
200 000 cells (Kato-, CHO/17-1A- or CHO-cells) were incubated with
one of the periplasma preparations containing relevant or
irrelevant Fab, followed by biotinylated polyclonale
anti-human-kappa light chain antibody (20 .mu.g/ml PBS) and
FITC-conjugated Streptavidine. Labeled cells were then analyzed by
flow cytometry. The periplasma preparations containing the
k8-D4.5-Fab and the k5.1-D4.5-Fab, respectively showed distinct
signals compared to irrelevant periplasma preparation (negative
control), but no staining of untransfected CHO-cells thus
demonstrating specificity for the 17-1A-antigen. The anti 17-1A
antibody M79 (Gottlinger, Int. J. Cancer 38 (1986), 47 53) was used
as a positive control for 17-1A-positive cells and a murine IgG2a
antibody as isotype control (see FIGS. 10 and 11).
EXAMPLE III
Eucaryotic Expression in CHO-Cells
Bacteria are usually not capable of producing complete functional
immunoglobulins although they express functional Fab fragments.
For the production of complete functional antibodies, mammalian
cells have to be used and therefore the k8-light chain and the
variable domain of D4.5 heavy chain were subcloned into mammalian
expression vectors.
a.) light chains (k8 and k5.1): To generate suitable terminal
restriction sites, the k8 and the k5.1 DNA fragments were
reamplified by PCR, resulting in kappa fragments with a Bsu36I-site
at the 5'-end as well as a Sal I and a Not I-site at the
3'-end.
These fragments (k8 and k5.1) were subcloned into the plasmid
BSPOLL by Bsu36I and Not I, thus adding a mammalian leader sequence
and sequenzed for preventing PCR-induced mutations.
Utilizing EcoRI and SalI, k8 and k5.1 were excised from BSPOLL and
subcloned into the eucaryotic expression vector pEF-ADA (see FIG.
12) derived from the expression vector pEF-DHFR (Mack, Proc. Natl.
Acad. Sci. U.S.A. 92 (1995) 7021 7025) by replacing the cDNA
encoding murine dihydrofolate reductase (DHFR) by that encoding
murine adenosine deaminase (ADA).
For each light chain species (k8 and k5.1), 10.sup.7 CHO cells were
transfected with 100 .mu.g of linearized plasmid DNA, respectively
and subsequently cultured under conditions selecting for adenosine
desaminase (ADA) activity encoded by the expression vector.
Surviving ADA-positive cells were cultured for further transfection
with heavy chains carrying the D4.5- or the D7.2-variable
domain.
b.) heavy 4.5 variable domain: From the delta Fd-fragment D4.5, the
variable region was reamplified by PCR generating Bsu36I
restriction sites at both ends.
The resulting V-D4.5 DNA-fragment was then subcloned by using these
restriction sites, into the eucaryotic expression vector pEF-DHFR
already containing an eucaryotic leader sequence as well as a
DNA-fragment encoding the human IgG1 heavy chain constant region
(see FIG. 13). The D4.5 heavy chain variable region was thus
inserted between the leader and the heavy chain constant
region.
The variable region was sequenced and the complete clone was
designated H79V-D4.5 hu IgG1.
For later tissue staining, the human IgG1 heavy chain constant
region was replaced by the murine IgG1 heavy chain constant region
using Xba I for subcloning. This plasmid was designated H79V-D4.5
MIgG1.
Both, the human and the murine IgG1-version of the D4.5 heavy chain
were each transfected into 10.sup.7 CHO-cells, already expressing
the k8 light chain, respectively. The human IgG1-version was also
transfected into CHO cells expressing the k5.1 light chain. 100
.mu.g linearized plasmid DNA was used for heavy chain transfection,
respectively.
The variable region of the D7.2 heavy chain Fd-fragment was also
subcloned into pEF-DHFR resulting in a human IgG1 heavy chain
expressing plasmid as described for VD4.5. This expression plasmid
was then transfected into CHO-cells already expressing the k8 light
chain as described above for H79V-D4.5 hu IgG1. The transfected
cells were subjected to selection for ADA- and DHFR-activity as
described (Kaufman, Methods Enzymol. 185 (1990), 537 566).
The resulting cell lines were designated H79-huIgG1
(VD4.5huIgG1-k8), H79-MIgG1 (VD4.5MIgG1-k8), D7.2 (VD7.2huIgG1-k8)
and HD70 (VD4.5huIgG1-k5.1).
Three days old culture supernatants from four different confluent
30 ml cell-cultures each producing one of the four
anti-17-1A-antibodies (H79-huIgG1, H79-MIgG1, D7.2 and HD70) were
tested by ELISA for binding to immobilized s17-1A. Except for D7.2
showing a weak ELISA-signal, the three other antibodies showed
strong signals, estimated to represent a binding affinity in the
range of the murine antibody M79.
Large scale antibody production was carried out in rollerbottles
using 500 ml medium.
The antibodies H79-huIgG1, D7.2 and HD70 were purified by using a
protein A affinity column. The H79-MIgG1 antibody was purified by
anti-mouse IgG affinity chromatography.
Purity and molecular weight of the recombinant antibodies were
determined by SDS-PAGE under reducing and nonreducing conditions
(FIGS. 14 and 15).
Protein purification and SDS-PAGE were carried out according to
standard procedures.
EXAMPLE IV
Functional Analysis of the H79 Antibodies and HD70
IV.1. Test on Immobilized Antigen
Three days old culture supernatant of a confluent 30 ml culture of
human and murine IgG1-transfectants respectively as well as the
corresponding preparations of purified antibody were tested for
binding on immobilized s17-1A antigen by ELISA and compared to the
murine M79 anti 17-1A antibody.
Detection was carried out as described in II.
The antibodies H79 (huIgG1- and MIgG1 version) and HD70 were
estimated to have very similar binding affinities in the range of
the murine M79.
IV.2. Determination of Affinities
Surface plasmon resonance measurement was performed using the
BIACORE 2000 device (Biacore AB, Freiburg, Freiburg, Germany).
Immobilization of recombinant soluble 17-1A-antigen in each flow
cell and analysis of the interaction was carried out with an
automatic method in BIACORE 2000. The antigen was covalently
coupled to sensor chip CM5 via primary amine groups. After
activation of the carboxylated dextran matrix of CM5 sensor chip
with a single injection of 80 .mu.l of 0.1 M
N-hydroxisuccinimide/0.4 M
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide (NHS/EDC9) through
all four flow cells, flow cells 1, 2, 3 and 4 were successively
included ito the flow path during injection of s17-1A-antigen (60
.mu.g/ml in 10 mM sodium acetate, pH 4.7). Different contact times
of 17-1A to the activated surface lead to approximately 2500
response units (RU9 in flow cell 1, 14000 RU in flow cell 2, 780 RU
in flow cell 3 and 290 RU in flow cell 4). Excess activated esters
were blocked by injection of 85 .mu.l 1 M ethanolamine pH 5 over
all four flow cells. Binding experiments were performed at
25.degree. C. in buffer of pH 7.4 containing 10 mM Hepes, 150 mM
NaCl, 3 mM EDTA and 0.005% surfactant P20. The binding kinetics of
antibodies to immobilized recombinant 17-1A were determined by
injecting antibody concentrations ranging from 0.5 to 2 .mu.M. The
sensor chip was regenerated between each run with 100 mM Glycin,
500 mM NaCl, 0.005% Tween pH3. The association and dissociation
rate constants, Kon and Koff were analysed using BIA-evaluation
software from Biacore AB as described (Karlsson, (1991) J Immunol
Meth 145: 229 240).
Two sets of affinity determinations were carried out (1. set: M79,
H79, D7.2, Panorex; 2. set: Panorex, HD70); the murine Panorex was
also included in set 2 as al reference.
The K.sub.D of D7.2 proved to be below the minimal detection value
of 10.sup.-4 M and is fore not shown in Tab 2.
TABLE-US-00002 TABLE 2 K.sub.D, K.sub.on and K.sub.off -rates of
human and relevant murine 17-1A antibodies K.sub.on K.sub.off
K.sub.D Antibody (M.sup.-1s.sup.-1) (s.sup.-1) (M) 1. set: murine
M79 6.0 .times. 10.sup.4 4.5 .times. 10.sup.-2 7.5 .times.
10.sup.-7 human H79 2.1 .times. 10.sup.4 7.2 .times. 10.sup.-3 3.4
.times. 10.sup.-7 murine Panorex 1.1 .times. 10.sup.5 2.2 .times.
10.sup.-2 2.0 .times. 10.sup.-7 2. set: human HD70 0.9 .times.
10.sup.5 3.5 .times. 10.sup.-2 3.9 .times. 10.sup.-7 murine Panorex
1.0 .times. 10.sup.5 2.7 .times. 10.sup.-2 2.7 .times.
10.sup.-7
IV.3. Flow Cytometry on 17-1A Expressing Eucaryotic Cells
Purified antibody preparations of H79 (human and murine IgG1
version), HD70 (human IgG1) and D7.2 (human IgG1) were tested by
FACS analysis on 17-1A expressing Kato cells, 17-1A-transfected
CHO-cells and untransfected CHO-cells. 2.times.10.sup.5 cells were
incubated with purified H79-huIgG1, H79-MIgG1, HD70, D7.2, M79,
Panorex, M74ch (=human CHIgG1-human Ckappa-version of the murine
anti-17-1A-antibody M74 (Gottlinger, Int. J. Cancer 38 (1986), 47
53)), murine IgG2a, murine IgG1, or human IgG1 (20 .mu.g/ml
antibody each), respectively. Detection of cell-bound antibodies
was carried out with FITC labeled anti mouse IgG- or anti human IgG
antibodies (20 .mu.g/ml each). Incubation was carried out for 45 60
min. on ice.
H79 human IgG1, H79 murine IgG1 and HD70 showed distinct binding to
the 17-1A positive cells as well as M79 and Panorex. D7.2 showed
weak but significant binding to 17-1A positive cells. None of the
antibodies showed binding to untransfected CHO cells and
IgG-controls were negative on Kato-cells, CHO/17-1A-cells and
untransfected CHO cells (see FIGS. 16 and 17).
IV.4. Antibody Dependent Cellular Cytotoxicity (.sup.51Cr
Release)
For .sup.51Cr release, human peripheral blood mononuclear cells
(PBMCs) as effector cells were isolated from the fresh buffy coat
of healthy donors. The PBMCs were separated by Ficoll density
gradient centrifugation with a subsequent 100.times.g
centrifugation step. Unstimulated PBMCs (5.times.10.sup.5 cells)
were added in a volume of 100 .mu.l of RPMI 1640 medium with 10%
FCS to each well of a flatbottomed microtiter plate and incubated
overnight at 37.degree. C. Target cells were labelled for 2 h with
.sup.51Cr. Labeled target cells (100 .mu.l) and antibodies in
different concentrations (50 .mu.l) were added to the PBMCs and
incubated for 18 h at 37.degree. C. Corresponding non-binding
isotypes were used as negative controls. Specific lysis was
calculated as ((cpm, experimental release)-(cpm, spontaneous
release))/((cpm, maximal release)-(cpm, spontaneous release)).
The human anti 17-1A antibodies H79 and HD70 proved to mediate high
cytotoxicity for the 17-1A positive gastric cancer cell line KATO
in this .sup.51Cr release assay. The murine anti 17-1A antibodies
M79 and Panorex proved to mediate cell killing to a distinctly
lower level (FIG. 18).
IV.5. Test on Human Tissue
5 nm cryosections of a colon carcinoma and normal colon tissue
respectively were incubated with the murine IgG-version of the
antibody H79 (10 .mu.g/ml). In this experiment the murine IgG1
version of H79 was used to avoid unspecific staining due to the
presence of human immunoglobulin in human tissue. Detection of
bound H79MIgG1 was carried out with a peroxidase conjugated
polyclonal anti mouse Ig antibody and stained with carbazole.
Counter staining was carried out with hemalaun.
Results were evaluated by light microscopy.
H79MIgG1 as well as the murine monoclonal antibody M79 (positive
control) showed strong staining on normal colon mucosa (M79, FIG.
19; H79-MIgG1, FIG. 20) and weaker staining on colon carcinoma
cells (M79, FIG. 21; H79-MIgG1, FIG. 22). In contrast, Isotype
controls showed no staining on colon mucosa and colon carcinoma
tissues (for M79; FIGS. 23 and 25 and for H79-MIgG1, FIGS. 24 and
26).
EXAMPLE V
Epitope Analysis of H79 and HD70
To compare the 17-1A-epitopes recognized by the human antibodies
H79 and HD70, and by the murine template antibody M79, 13mer
peptides derived from the amino acid sequence of the extracellular
part of the 17-1A-antigen were sythesized as single spots on a Pep
Spots Membrane. These peptides cover the whole extracellular amino
acid sequence of the 17-1A-antigen (as defined by Szala, Proc.
Natl. Acad. Sci. U.S.A. 87 (1990), 3542 3546) by having an overlap
of 11 amino acids with each of the neighboring peptides with the
first amino acid of peptide 1 being identical with the N-terminal
amino acid of the 17-1A antigen and the last amino acid of peptide
119 being identical with the C-terminal amino acid of the
extracellular part of the 17-1A-antigen (Tab. 3).
TABLE-US-00003 TABLE 3 Synthesized peptides (numbers correspond to
the numbers of peptide spots) 1. TATFAAAQEECVC SEQ ID NO.:22 2.
TFAAAQEECVCEN SEQ ID NO.:23 3. AAAQEECVCENYK SEQ ID NO.:24 4.
AQEECVCENYKLA SEQ ID NO.:25 5. EECVCENYKLAVN SEQ ID NO.:26 6.
CVCENYKLAVNCF SEQ ID NO.:27 7. CENYKLAVNCFVN SEQ ID NO.:28 8.
NYKLAVNCFVNNN SEQ ID NO.:29 9. KLAVNCFVNNNRQ SEQ ID NO.:30 10.
AVNCFVNNNRQCQ SEQ ID NO.:31 11. NCFVNNNRQCQCT SEQ ID NO.:32 12.
FVNNNRQCQCTSV SEQ ID NO.:33 13. NNNRQCQCTSVGA SEQ ID NO.:34 14.
NRQCQCTSVGAQN SEQ ID NO.:35 15. QCQCTSVGAQNTV SEQ ID NO.:36 16.
QCTSVGAQNTVIC SEQ ID NO.:37 17. TSVGAQNTVICSK SEQ ID NO.:38 18.
VGAQNTVICSKLA SEQ ID NO.:39 19. AQNTVICSKLAAK SEQ ID NO.:40 20.
NTVICSKLAAKCL SEQ ID NO.:41 21. VICSKLAAKCLVM SEQ ID NO.:42 22.
CSKLAAKCLVMKA SEQ ID NO.:43 23. KLAAKCLVMKAEM SEQ ID NO.:44 24.
AAKCLVMKAEMNG SEQ ID NO.:45 25. KCLVMKAEMNGSK SEQ ID NO.:46 26.
LVMKAEMNGSKLG SEQ ID NO.:47 27. MKAEMNGSKLGRR SEQ ID NO.:48 28.
AEMNGSKLGRRAK SEQ ID NO.:49 29. MNGSKLGRRAKPE SEQ ID NO.:50 30.
GSKLGRRAKPEGA SEQ ID NO.:51 31. KLGRRAKPEGALQ SEQ ID NO.:52 32.
GRRAKPEGALQNN SEQ ID NO.:53 33. RAKPEGALQNNDG SEQ ID NO.:54 34.
KPEGALQNNDGLY SEQ ID NO.:55 35. EGALQNNDGLYDP SEQ ID NO.:56 36.
ALQNNDGLYDPDC SEQ ID NO.:57 37. QNNDGLYDPDCDE SEQ ID NO.:58 38.
NDGLYDPDCDESG SEQ ID NO.:59 39. GLYDPDCDESGLF SEQ ID NO.:60 40.
YDPDCDESGLFKA SEQ ID NO.:61 41. PDCDESGLFKAKQ SEQ ID NO.:62 42.
CDESGLFKAKQCN SEQ ID NO.:63 43. ESGLFKAKQCNGT SEQ ID NO.:64 44.
GLFKAKQCNGTST SEQ ID NO.:65 45. FKAKQCNGTSTCW SEQ ID NO.:66 46.
AKQCNGTSTCWCV SEQ ID NO.:67 47. QCNGTSTCWCVNT SEQ ID NO.:68 48.
NGTSTCWCVNTAG SEQ ID NO.:69 49. TSTCWCVNTAGVR SEQ ID NO.:70 50.
TCWCVNTAGVRRT SEQ ID NO.:71 51. WCVNTAGVRRTDK SEQ ID NO.:72 52.
VNTAGVRRTDKDT SEQ ID NO.:73 53. TAGVRRTDKDTEI SEQ ID NO.:74 54.
GVRRTDKDTEITC SEQ ID NO.:75 55. RRTDKDTEITCSE SEQ ID NO.:76 56.
TDKDTEITCSERV SEQ ID NO.:77 57. KDTEITCSERVRT SEQ ID NO.:78 58.
TEITCSERVRTYW SEQ ID NO.:79 59. ITCSERVRTYWII SEQ ID NO.:80 60.
CSERVRTYWIIIE SEQ ID NO.:81 61. ERVRTYWIIIELK SEQ ID NO.:82 62.
VRTYWIIIELKHK SEQ ID NO.:83 63. TYWIIIELKHKAR SEQ ID NO.:84 64.
WIIIELKHKAREK SEQ ID NO.:85 65. IIELKHKAREKPY SEQ ID NO.:86 66.
ELKHKAREKPYDS SEQ ID NO.:87 67. KHKAREKPYDSKS SEQ ID NO.:88 68.
KAREKPYDSKSLR SEQ ID NO.:89 69. REKPYDSKSLRTA SEQ ID NO.:90 70.
KPYDSKSLRTALQ SEQ ID NO.:91 71. YDSKSLRTALQKE SEQ ID NO.:92 72.
SKSLRTALQKEIT SEQ ID NO.:93 73. SLRTALQKEITTR SEQ ID NO.:94 74.
RTALQKEITTRYQ SEQ ID NO.:95 75. ALQKEITTRYQLD SEQ ID NO.:96 76.
QKEITTRYQLDPK SEQ ID NO.:97 77. EITTRYQLDPKFI SEQ ID NO.:98 78.
TTRYQLDPKFITS SEQ ID NO.:99 79. RYQLDPKFITSIL SEQ ID NO.:100 80.
QLDPKFITSILYE SEQ ID NO.:101 81. DPKFITSILYENN SEQ ID NO.:102 82.
KFITSILYENNVI SEQ ID NO.:103 83. ITSILYENNVITI SEQ ID NO.:104 84.
SILYENNVITIDL SEQ ID NO.:105 85. LYENNVITIDLVQ SEQ ID NO.:106 86.
ENNVITIDLVQNS SEQ ID NO.:107 87. NVITIDLVQNSSQ SEQ ID NO.:108 88.
ITIDLVQNSSQKT SEQ ID NO.:109 89. IDLVQNSSQKTQN SEQ ID NO.:110 90.
LVQNSSQKTQNDV SEQ ID NO.:111 91. QNSSQKTQNDVDI SEQ ID NO.:112 92.
SSQKTQNDVDIAD SEQ ID NO.:113 93. QKTQNDVDIADVA SEQ ID NO.:114 94.
TQNDVDIADVAYY SEQ ID NO.:115 95. NDVDIADVAYYFE SEQ ID NO.:116 96.
VDIADVAYYFEKD SEQ ID NO.:117 97. IADVAYYFEKDVK SEQ ID NO.:118 98.
DVAYYFEKDVKGE SEQ ID NO.:119 99. AYYFEKDVKGESL SEQ ID NO.:120 100.
YFEKDVKGESLFH SEQ ID NO.:121 101. EKDVKGESLFHSK SEQ ID NO.:122 102.
DVKGESLFHSKKM SEQ ID NO.:123 103. KGESLFHSKKMDL SEQ ID NO.:124 104.
ESLFHSKKMDLTV SEQ ID NO.:125 105. LFHSKKMDLTVNG SEQ ID NO.:126 106.
HSKKMDLTVNGEQ SEQ ID NO.:127 107. KKMDLTVNGEQLD SEQ ID NO.:128 108.
MDLTVNGEQLDLD SEQ ID NO.:129 109. LTVNGEQLDLDPG SEQ ID NO.:130 110.
VNGEQLDLDPGQT SEQ ID NO.:131 111. GEQLDLDPGQTLI SEQ ID NO.:132 112.
QLDLDPGQTLIYY SEQ ID NO.:133 113. DLDPGQTLIYYVD SEQ ID NO.:134 114.
DPGQTLIYYVDEK SEQ ID NO.:135 115. GQTLIYYVDEKAP SEQ ID NO.:136 116.
TLIYYVDEKAPEF SEQ ID NO.:137 117. IYYVDEKAPEFSM SEQ ID NO.:138 118.
YVDEKAPEFSMQG SEQ ID NO.:139 119. DEKAPEFSMQGLK SEQ ID NO.:140
The PepSpots Membrane with the synthesized peptides was shaken for
ten minutes in methanol and then washed three times for ten minutes
in TBS-buffer pH8. The membrane was then blocked in casein based
blocking solution, containing 0.05 g/ml sucrose for one hour and
then once shaken in TBS pH8/0.05% Tween 20 (TBS-T). Each of the
anti-17-1A antibodies was incubated at room temperature together
with the membrane for three hours in blocking solution (1 .mu.g
antibody/ml). After three washes in TBS-T for ten minutes each, the
horse radish peroxidase (HRP)-conjugated secondary antibody (anti
mouse/anti human; 1 .mu.g/ml blocking solution) was incubated for
two hours at room temperature together with the membrane. Then the
membrane was washed for three times in TBS-T for ten minutes each.
Detection of bound antibodies was performed directly on the Pep
Spots Membrane in case of M79 according to the protocol of the
chemoluminescence kit manufacturer (Boehringer). The developed
films are shown in FIG. 27. The blot was regenerated after three
washes with TBS-T (10 min.) in a solution containing 50 mM Tris-HCl
pH 6.7, 100 mM 2-Mercaptoethanol and 2% (w/v) SDS for 30 min at
50.degree. C. and subsequently reused for epitope mapping of the
next antibody.
Due to the higher degree of unspecific binding of the anti-human
immunoglobulin secondary antibody to the polypeptide-spots on the
membrane, a fractionated electrotransfer to a second
blotting-membrane followed by detection with the anti-human
secondary antibody was carried out in case of HD70 and H79
according to Rudiger, EMBO 16 (1997) 1501 1507. Incubation with the
secondary antibody and antibody detection on the blots was
performed as described above. The developed films are presented in
FIG. 27.
As shown in this figure (FIG. 27), the results of peptide based
epitope mapping indicate, that the epitope recognized by the murine
template antibody M79, mainly represented by peptide spots 38 and
95, profoundly differs from that recognized by the human antibody
H79, mainly represented by peptide spots 8, 11, 13, 14, 59 60, 77
and 79. As the human antibody HD70 shows no detectable binding at
all, it can be further anticipated, that its epitope also differs
from that of the murine antibody M79 and that the epitopes of the
human antibodies H79 and HD70 are not identical, too.
The absence of detectable binding signals in case of the human
antibody HD70 may be explained by its possible recognition either
of a conformational, continuous or discontinuous epitope or of an
epitope partially or entirely consisting of carbohydrate; in either
case mimicking of such epitopes by short peptides can hardly be
expected.
SEQUENCE LISTINGS
1
169122DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 1aggtgcagct gctcgagtct gg 22224DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
2cagagtcacc ttgctcgagt ctgg 24323DNAArtificial SequenceDescription
of Artificial Sequence Synthetic Primer 3caggtgcagc tgctcgagtc ggg
23423DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 4caggtgcagc tactcgagtg ggg 23523DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
5caggtacagc tgctcgagtc agg 23629DNAArtificial SequenceDescription
of Artificial Sequence Synthetic Primer 6tgccttactg tctctggcca
gcggaagat 29738DNAArtificial SequenceDescription of Artificial
Sequence Synthetic Primer 7gagccgcacg agcccgagct ccagatgacc
cagtctcc 38840DNAArtificial SequenceDescription of Artificial
Sequence Synthetic Primer 8gagccgcacg agcccgagct cgtgattgac
agcagtctcc 40939DNAArtificial SequenceDescription of Artificial
Sequence Synthetic Primer 9gagccgcacg agcccgagct cgtgatgacc
tcagtctcc 391038DNAArtificial SequenceDescription of Artificial
Sequence Synthetic Primer 10gagccgcacg agcccgagct cacactcacg
cagtctcc 381138DNAArtificial SequenceDescription of Artificial
Sequence Synthetic Primer 11gagccgcacg agcccgagct cgtgctgact
cagtctcc 381258DNAArtificial SequenceDescription of Artificial
Sequence Synthetic Primer 12gcgccgtcta gaattaacac tctcccctgt
tgaagctctt tgtgacgggc gaactcag 581324DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
13aattttgagc tcactcagcc ccac 241427DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
14tctgccgagc tccagcctgc ctccgtg 271527DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
15tctgtggagc tccagccgcc ctcagtg 271633DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
16tctgaagagc tccaggaccc tgttgtgtct gtg 331724DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
17cagtctgagc tcacgcagcc gccc 241824DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
18cagactgagc tcactcagga gccc 241924DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
19caggttgagc tcactcaacc gccc 242027DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
20caggctgagc tcactcagcc gtcttcc 272130DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
21cgccgtctag aattatgaac attctgtagg 302213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 22Thr
Ala Thr Phe Ala Ala Ala Gln Glu Glu Cys Val Cys 1 5
102313PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 23Thr Phe Ala Ala Ala Gln Glu Glu Cys Val Cys Glu
Asn 1 5 102413PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 24Ala Ala Ala Gln Glu Glu Cys Val Cys
Glu Asn Tyr Lys 1 5 102513PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 25Ala Gln Glu Glu Cys Val Cys
Glu Asn Tyr Lys Leu Ala 1 5 102613PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Peptide 26Glu Glu Cys Val Cys Glu
Asn Tyr Lys Leu Ala Val Asn 1 5 102713PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 27Cys
Val Cys Glu Asn Tyr Lys Leu Ala Val Asn Cys Phe 1 5
102813PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 28Cys Glu Asn Tyr Lys Leu Ala Val Asn Cys Phe Val
Asn 1 5 102913PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 29Asn Tyr Lys Leu Ala Val Asn Cys Phe
Val Asn Asn Asn 1 5 103013PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 30Lys Leu Ala Val Asn Cys Phe
Val Asn Asn Asn Arg Gln 1 5 103113PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Peptide 31Ala Val Asn Cys Phe Val
Asn Asn Asn Arg Gln Cys Gln 1 5 103213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 32Asn
Cys Phe Val Asn Asn Asn Arg Gln Cys Gln Cys Thr 1 5
103313PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 33Phe Val Asn Asn Asn Arg Gln Cys Gln Cys Thr Ser
Val 1 5 103413PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 34Asn Asn Asn Arg Gln Cys Gln Cys Thr
Ser Val Gly Ala 1 5 103513PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 35Asn Arg Gln Cys Gln Cys Thr
Ser Val Gly Ala Gln Asn 1 5 103613PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Peptide 36Gln Cys Gln Cys Thr Ser
Val Gly Ala Gln Asn Thr Val 1 5 103713PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 37Gln
Cys Thr Ser Val Gly Ala Gln Asn Thr Val Ile Cys 1 5
103813PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 38Thr Ser Val Gly Ala Gln Asn Thr Val Ile Cys Ser
Lys 1 5 103913PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 39Val Gly Ala Gln Asn Thr Val Ile Cys
Ser Lys Leu Ala 1 5 104013PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 40Ala Gln Asn Thr Val Ile Cys
Ser Lys Leu Ala Ala Lys 1 5 104113PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Peptide 41Asn Thr Val Ile Cys Ser
Lys Leu Ala Ala Lys Cys Leu 1 5 104213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 42Val
Ile Cys Ser Lys Leu Ala Ala Lys Cys Leu Val Met 1 5
104313PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 43Cys Ser Lys Leu Ala Ala Lys Cys Leu Val Met Lys
Ala 1 5 104413PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 44Lys Leu Ala Ala Lys Cys Leu Val Met
Lys Ala Glu Met 1 5 104512PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 45Ala Ala Lys Cys Leu Val Met
Lys Ala Glu Asn Gly 1 5 104613PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 46Lys Cys Leu Val Met Lys Ala
Glu Met Asn Gly Ser Lys 1 5 104713PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Peptide 47Leu Val Met Lys Ala Glu
Met Asn Gly Ser Lys Leu Gly 1 5 104813PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 48Met
Lys Ala Glu Met Asn Gly Ser Lys Leu Gly Arg Arg 1 5
104913PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 49Ala Glu Met Asn Gly Ser Lys Leu Gly Arg Arg Ala
Lys 1 5 105013PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 50Met Asn Gly Ser Lys Leu Gly Arg Arg
Ala Lys Pro Glu 1 5 105113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 51Gly Ser Lys Leu Gly Arg Arg
Ala Lys Pro Glu Gly Ala 1 5 105213PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Peptide 52Lys Leu Gly Arg Arg Ala
Lys Pro Glu Gly Ala Leu Gln 1 5 105313PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 53Gly
Arg Arg Ala Lys Pro Glu Gly Ala Leu Gln Asn Asn 1 5
105413PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 54Arg Ala Lys Pro Glu Gly Ala Leu Gln Asn Asn Asp
Gly 1 5 105513PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 55Lys Pro Glu Gly Ala Leu Gln Asn Asn
Asp Gly Leu Tyr 1 5 105613PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 56Glu Gly Ala Leu Gln Asn Asn
Asp Gly Leu Tyr Asp Pro 1 5 105713PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Peptide 57Ala Leu Gln Asn Asn Asp
Gly Leu Tyr Asp Pro Asp Cys 1 5 105813PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 58Gln
Asn Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Glu 1 5
105913PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 59Asn Asp Gly Leu Tyr Asp Pro Asp Cys Asp Glu Ser
Gly 1 5 106013PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 60Gly Leu Tyr Asp Pro Asp Cys Asp Glu
Ser Gly Leu Phe 1 5 106113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 61Tyr Asp Pro Asp Cys Asp Glu
Ser Gly Leu Phe Lys Ala 1 5 106213PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Peptide 62Pro Asp Cys Asp Glu Ser
Gly Leu Phe Lys Ala Lys Gln 1 5 106313PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 63Cys
Asp Glu Ser Gly Leu Phe Lys Ala Lys Gln Cys Asn 1 5
106413PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 64Glu Ser Gly Leu Phe Lys Ala Lys Gln Cys Asn Gly
Thr 1 5 106513PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 65Gly Leu Phe Lys Ala Lys Gln Cys Asn
Gly Thr Ser Thr 1 5 106613PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 66Phe Lys Ala Lys Gln Cys Asn
Gly Thr Ser Thr Cys Trp 1 5 106713PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Peptide 67Ala Lys Gln Cys Asn Gly
Thr Ser Thr Cys Trp Cys Val 1 5 106813PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 68Gln
Cys Asn Gly Thr Ser Thr Cys Trp Cys Val Asn Thr 1 5
106913PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 69Asn Gly Thr Ser Thr Cys Trp Cys Val Asn Thr Ala
Gly 1 5 107013PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 70Thr Ser Thr Cys Trp Cys Val Asn Thr
Ala Gly Val Arg 1 5 107113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 71Thr Cys Trp Cys Val Asn Thr
Ala Gly Val Arg Arg Thr 1 5 107213PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Peptide 72Trp Cys Val Asn Thr Ala
Gly Val Arg Arg Thr Asp Lys 1 5 107313PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 73Val
Asn Thr Ala Gly Val Arg Arg Thr Asp Lys Asp Thr 1 5
107413PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 74Thr Ala Gly Val Arg Arg Thr Asp Lys Asp Thr Glu
Ile 1 5 107513PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 75Gly Val Arg Arg Thr Asp Lys Asp Thr
Glu Ile Thr Cys 1 5 107613PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 76Arg Arg Thr Asp Lys Asp Thr
Glu Ile Thr Cys Ser Glu 1 5 107713PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Peptide 77Thr Asp Lys Asp Thr Glu
Ile Thr Cys Ser Glu Arg Val 1 5 107813PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 78Lys
Asp Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr 1 5
107913PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 79Thr Glu Ile Thr Cys Ser Glu Arg Val Arg Thr Tyr
Trp 1 5 108013PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 80Ile Thr Cys Ser Glu Arg Val Arg Thr
Tyr Trp Ile Ile 1 5 108113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 81Cys Ser Glu Arg Val Arg Thr
Tyr Trp Ile Ile Ile Glu 1 5 108213PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Peptide 82Glu Arg Val Arg Thr Tyr
Trp Ile Ile Ile Glu Leu Lys 1 5 108313PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 83Val
Arg Thr Tyr Trp Ile Ile Ile Glu Leu Lys His Lys 1 5
108413PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 84Thr Tyr Trp Ile Ile Ile Glu Leu Lys His Lys Ala
Arg 1 5 108513PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 85Trp Ile Ile Ile Glu Leu Lys His Lys
Ala Arg Glu Lys 1 5 108613PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 86Ile Ile Glu Leu Lys His Lys
Ala Arg Glu Lys Pro Tyr 1 5 108713PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Peptide 87Glu Leu Lys His Lys Ala
Arg Glu Lys Pro Tyr Asp Ser 1 5 108813PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 88Lys
His Lys Ala Arg Glu Lys Pro Tyr Asp Ser Lys Ser 1 5
108913PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 89Lys Ala Arg Glu Lys Pro Tyr Asp Ser Lys Ser Leu
Arg 1 5 109013PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 90Arg Glu Lys Pro Tyr Asp Ser Lys Ser
Leu Arg Thr Ala 1 5 109113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 91Lys Pro Tyr Asp Ser Lys Ser
Leu Arg Thr Ala Leu Gln 1 5 109213PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Peptide 92Tyr Asp Ser Lys Ser Leu
Arg Thr Ala Leu Gln Lys Glu 1 5 109313PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 93Ser
Lys Ser Leu Arg Thr Ala Leu Gln Lys Glu Ile Thr 1 5
109413PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 94Ser Leu Arg Thr Ala Leu Gln Lys Glu Ile Thr Thr
Arg 1 5 109513PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 95Arg Thr Ala Leu Gln Lys Glu Ile Thr
Thr Arg Tyr Gln 1 5 109613PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 96Ala Leu Gln Lys Glu Ile Thr
Thr Arg Tyr Gln Leu Asp 1 5 109713PRTArtificial SequenceDescription
of Artificial Sequence Synthetic Peptide 97Gln Lys Glu Ile Thr Thr
Arg Tyr Gln Leu Asp Pro Lys 1 5 109813PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 98Glu
Ile Thr Thr Arg Tyr Gln Leu Asp Pro Lys Phe Ile 1 5
109913PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 99Thr Thr Arg Tyr Gln Leu Asp Pro Lys Phe Ile Thr
Ser 1 5 1010013PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 100Arg Tyr Gln Leu Asp Pro Lys Phe Ile
Thr Ser Ile Leu 1 5 1010113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 101Gln Leu Asp Pro Lys Phe
Ile Thr Ser Ile Leu Tyr Glu 1 5 1010213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 102Asp
Pro Lys Phe Ile Thr Ser Ile Leu Tyr Glu Asn Asn 1 5
1010313PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 103Lys Phe Ile Thr Ser Ile Leu Tyr Glu Asn
Asn
Val Ile 1 5 1010413PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 104Ile Thr Ser Ile Leu Tyr Glu Asn Asn
Val Ile Thr Ile 1 5 1010513PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 105Ser Ile Leu Tyr Glu Asn
Asn Val Ile Thr Ile Asp Leu 1 5 1010613PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 106Leu
Tyr Glu Asn Asn Val Ile Thr Ile Asp Leu Val Gln 1 5
1010713PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 107Glu Asn Asn Val Ile Thr Ile Asp Leu Val Gln
Asn Ser 1 5 1010813PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 108Asn Val Ile Thr Ile Asp Leu Val Gln
Asn Ser Ser Gln 1 5 1010913PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 109Ile Thr Ile Asp Leu Val
Gln Asn Ser Ser Gln Lys Thr 1 5 1011013PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 110Ile
Asp Leu Val Gln Asn Ser Ser Gln Lys Thr Gln Asn 1 5
1011113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 111Leu Val Gln Asn Ser Ser Gln Lys Thr Gln Asn
Asp Val 1 5 1011213PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 112Gln Asn Ser Ser Gln Lys Thr Gln Asn
Asp Val Asp Ile 1 5 1011313PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 113Ser Ser Gln Lys Thr Gln
Asn Asp Val Asp Ile Ala Asp 1 5 1011413PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 114Gln
Lys Thr Gln Asn Asp Val Asp Ile Ala Asp Val Ala 1 5
1011513PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 115Thr Gln Asn Asp Val Asp Ile Ala Asp Val Ala
Tyr Tyr 1 5 1011613PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 116Asn Asp Val Asp Ile Ala Asp Val Ala
Tyr Tyr Phe Glu 1 5 1011713PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 117Val Asp Ile Ala Asp Val
Ala Tyr Tyr Phe Glu Lys Asp 1 5 1011813PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 118Ile
Ala Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys 1 5
1011913PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 119Asp Val Ala Tyr Tyr Phe Glu Lys Asp Val Lys
Gly Glu 1 5 1012013PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 120Ala Tyr Tyr Phe Glu Lys Asp Val Lys
Gly Glu Ser Leu 1 5 1012113PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 121Tyr Phe Glu Lys Asp Val
Lys Gly Glu Ser Leu Phe His 1 5 1012213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 122Glu
Lys Asp Val Lys Gly Glu Ser Leu Phe His Ser Lys 1 5
1012313PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 123Asp Val Lys Gly Glu Ser Leu Phe His Ser Lys
Lys Met 1 5 1012413PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 124Lys Gly Glu Ser Leu Phe His Ser Lys
Lys Met Asp Leu 1 5 1012513PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 125Glu Ser Leu Phe His Ser
Lys Lys Met Asp Leu Thr Val 1 5 1012613PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 126Leu
Phe His Ser Lys Lys Met Asp Leu Thr Val Asn Gly 1 5
1012713PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 127His Ser Lys Lys Met Asp Leu Thr Val Asn Gly
Glu Gln 1 5 1012813PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 128Lys Lys Met Asp Leu Thr Val Asn Gly
Glu Gln Leu Asp 1 5 1012913PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 129Met Asp Leu Thr Val Asn
Gly Glu Gln Leu Asp Leu Asp 1 5 1013013PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 130Leu
Thr Val Asn Gly Glu Gln Leu Asp Leu Asp Pro Gly 1 5
1013113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 131Val Asn Gly Glu Gln Leu Asp Leu Asp Pro Gly
Gln Thr 1 5 1013213PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 132Gly Glu Gln Leu Asp Leu Asp Pro Gly
Gln Thr Leu Ile 1 5 1013313PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 133Gln Leu Asp Leu Asp Pro
Gly Gln Thr Leu Ile Tyr Tyr 1 5 1013413PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 134Asp
Leu Asp Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp 1 5
1013513PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 135Asp Pro Gly Gln Thr Leu Ile Tyr Tyr Val Asp
Glu Lys 1 5 1013613PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 136Gly Gln Thr Leu Ile Tyr Tyr Val Asp
Glu Lys Ala Pro 1 5 1013713PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 137Thr Leu Ile Tyr Tyr Val
Asp Glu Lys Ala Pro Glu Phe 1 5 1013813PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 138Ile
Tyr Tyr Val Asp Glu Lys Ala Pro Glu Phe Ser Met 1 5
1013913PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 139Tyr Val Asp Glu Lys Ala Pro Glu Phe Ser Met
Gln Gly 1 5 1014013PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 140Asp Glu Lys Ala Pro Glu Phe Ser Met
Gln Gly Leu Lys 1 5 10141321DNAArtificial SequenceDescription of
Artificial Sequence Synthetic Primer 141gag ctc cag atg acc cag tct
cca tcc tcc ctg tct gct tct gtg gga 48Glu Leu Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15gac aga gtc acc atc
act tgt cgg aca agt cag agc att agc agc tat 96Asp Arg Val Thr Ile
Thr Cys Arg Thr Ser Gln Ser Ile Ser Ser Tyr 20 25 30tta aat tgg tat
cag cag aaa cca gga cag cct cct aag ctg ctc att 144Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45tac tgg gca
tct acc cgg gaa tcc ggg gtc cct gac cga ttc agt ggc 192Tyr Trp Ala
Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60agc ggg
tct ggg aca gat ttc act ctc acc atc agc agt cta caa cct 240Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75
80gaa gat tct gca act tac tac tgt cag cag agt tac gac atc ccg tac
288Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Asp Ile Pro Tyr
85 90 95act ttt ggc cag ggg acc aag ctg gag atc aaa 321Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys 100 105142107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 142Glu
Leu Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10
15Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Ser Ile Ser Ser Tyr
20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu
Ile 35 40 45Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe
Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro 65 70 75 80Glu Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser
Tyr Asp Ile Pro Tyr 85 90 95Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile
Lys 100 105143414DNAArtificial SequenceDescription of Artificial
Sequence Synthetic Primer 143gag gtg cag ctg ctc gag tct ggg gga
ggc gtg gtc cag cct ggg agg 48Glu Val Gln Leu Leu Glu Ser Gly Gly
Gly Val Val Gln Pro Gly Arg 1 5 10 15tcc ctg aga ctc tcc tgt gca
gcc tct gga ttc acc ttc agt agc tat 96Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30ggc atg cac tgg gtc cgc
cag gct cca ggc aag ggg ctg gag tgg gtg 144Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45gca gtt ata tca tat
gat gga agt aat aaa tac tat gca gac tcc gtg 192Ala Val Ile Ser Tyr
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60aag ggc cga ttc
acc atc tcc aga gac aat tcc aag aac acg ctg tat 240Lys Gly Arg Phe
Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80ctg caa
atg aac agc ctg aga gct gag gac acg gct gtg tat tac tgt 288Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95gcg
aaa gat atg ggg tgg ggc agt ggc tgg aga ccc tac tac tac tac 336Ala
Lys Asp Met Gly Trp Gly Ser Gly Trp Arg Pro Tyr Tyr Tyr Tyr 100 105
110ggt atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tcc tca gca
384Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala
115 120 125ccc acc aag gct ccg gat gtg ttc cct cta 414Pro Thr Lys
Ala Pro Asp Val Phe Pro Leu 130 135144138PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 144Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Lys Asp Met Gly Trp Gly Ser Gly Trp
Arg Pro Tyr Tyr Tyr Tyr 100 105 110Gly Met Asp Val Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Ser Ala 115 120 125Pro Thr Lys Ala Pro Asp
Val Phe Pro Leu 130 135145372DNAArtificial SequenceDescription of
Artificial Sequence Synthetic Primer 145gag gtg cag ctg ctc gag tct
ggg gga gtc gtg gta cag cct ggg ggg 48Glu Val Gln Leu Leu Glu Ser
Gly Gly Val Val Val Gln Pro Gly Gly 1 5 10 15tcc ctg aga ctc tcc
tgt gca gcc tct gga ttc acc ttt gat gat tat 96Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30gcc atg cac tgg
gtc cgc cag gct cca ggc aag ggg ctg gag tgg gtg 144Ala Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45gca gtt ata
tca tat gat gga agt aat aaa tac tat gca gac tcc gtg 192Ala Val Ile
Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60aag ggc
cga ttc acc atc tcc aga gac aat tcc aag aac acg ctg tat 240Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75
80ctg caa atg aac agc ctg aga gct gag gac acg gct gtg tat tac tgt
288Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95gcg aaa aag gaa ggc tac tgg ggc cag gga acc ctg gtc acc gtc
tcc 336Ala Lys Lys Glu Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser 100 105 110tca gca ccc acc aag gct ccg gat gtg ttc cct cta
372Ser Ala Pro Thr Lys Ala Pro Asp Val Phe Pro Leu 115
120146124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 146Glu Val Gln Leu Leu Glu Ser Gly Gly Val Val
Val Gln Pro Gly Gly 1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Asp Asp Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Ser Tyr Asp Gly
Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Lys
Glu Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110Ser
Ala Pro Thr Lys Ala Pro Asp Val Phe Pro Leu 115
120147321DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 147gag ctc cag atg acc cag tct cca tcc tcc ctg tct
gca tct gta gga 48Glu Leu Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15gac aga gtc acc atc act tgc cgg gca agt
cag agc att agc agc tat 96Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Ser Ile Ser Ser Tyr 20 25 30tta aat tgg tat cag cag aaa cca gga
cag cct cct aag ctg ctc att 144Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Gln Pro Pro Lys Leu Leu Ile 35 40 45tac tgg gca tct acc cgg gaa tcc
ggg gtc cct gac cga ttc agc ggc 192Tyr Trp Ala Ser Thr Arg Glu Ser
Gly Val Pro Asp Arg Phe Ser Gly 50 55 60agt gaa tct ggg aca aat tac
act ctc acc atc agc agc ctg cag cct 240Ser Glu Ser Gly Thr Asn Tyr
Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80gaa gat ttt gct act
tac ttt tgt caa cag tct gac agt ttg ccg atc 288Glu Asp Phe Ala Thr
Tyr Phe Cys Gln Gln Ser Asp Ser Leu Pro Ile 85 90 95acc ttc ggc caa
ggg aca cga ctg gac att caa 321Thr Phe Gly Gln Gly Thr Arg Leu Asp
Ile Gln 100 105148107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 148Glu Leu Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile 35 40 45Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly 50 55 60Ser
Glu Ser Gly Thr Asn Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70
75 80Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Asp Ser Leu Pro
Ile 85 90 95Thr Phe Gly Gln Gly Thr Arg Leu Asp Ile Gln 100
10514922DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 149aggtgcagct gctcgagtct gg 2215024DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
150cagagtcacc ttgctcgagt ctgg 2415123DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
151caggtgcagc tgctcgagtc ggg 2315223DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
152caggtgcagc tactcgagtg ggg 2315323DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
153caggtacagc tgctcgagtc agg 2315429DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
154tgccttactg tctctggcca gcggaagat 2915538DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
155gagccgcacg agcccgagct ccagatgacc cagtctcc 3815640DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
156gagccgcacg agcccgagct cgtgattgac agcagtctcc 4015739DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
157gagccgcacg agcccgagct cgtgatgacc tcagtctcc
3915838DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 158gagccgcacg agcccgagct cacactcacg cagtctcc
3815938DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 159gagccgcacg agcccgagct cgtgctgact cagtctcc
3816058DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 160gcgccgtcta gaattaacac tctcccctgt tgaagctctt
tgtgacgggc gaactcag 5816124DNAArtificial SequenceDescription of
Artificial Sequence Synthetic Primer 161aattttgagc tcactcagcc ccac
2416227DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 162tctgccgagc tccagcctgc ctccgtg
2716327DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 163tctgtggagc tccagccgcc ctcagtg
2716433DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 164tctgaagagc tccaggaccc tgttgtgtct gtg
3316524DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 165cagtctgagc tcacgcagcc gccc 2416624DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
166cagactgagc tcactcagga gccc 2416724DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
167caggttgagc tcactcaacc gccc 2416827DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
168caggctgagc tcactcagcc gtcttcc 2716930DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
169cgccgtctag aattatgaac attctgtagg 30
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